Thyrotropin releasing hormone receptor-orexin receptor hetero-dimers/-oligomers

ABSTRACT

A hetero-dimeric or hetero-oligomeric receptor, comprising at least one thyrotropin releasing hormone receptor subunit associated with at least one orexin receptor subunit.

FIELD OF THE INVENTION

The present invention relates to a hetero-dimeric or hetero-oligomericreceptor, comprising at least one thyrotropin releasing hormone receptorsubunit associated with at least one orexin receptor subunit.

BACKGROUND ART

Proteins do not act in isolation in a cell, but in stable or transitorycomplexes, with protein-protein interactions being key determinants ofprotein function (Auerbach et al., (2002), Proteomics, 2, 611-623).Furthermore, proteins and protein complexes interact with other cellularcomponents like DNA, RNA and small molecules. Understanding both theindividual proteins involved in these interactions and theirinteractions are important for a better understanding of biologicalprocesses.

The functions of thyrotropin-releasing hormone (TRH) in the centralnervous system (CNS) are reported by Gary (Gary, Keith A., et al., TheThyrotropin-Releasing Hormone (TRH) Hypothesis of HomeostaticRegulation: Implications for TRH-Based Therapeutics, JPET 305:410-416,2003) as four anatomically distinct components that together comprise ageneral TRH homeostatic system, being 1) thehypothalamic-hypophysiotropic neuroendocrine system, 2) thebrainstem/midbrain/spinal cord system, 3) the limbic/cortical system,and 4) the chronobiological system.

Gary further notes that “an appreciation of the global function of TRHto modulate and normalize CNS activity, along with an appreciation ofthe inherent limitations of TRH itself as a therapeutic agent, leads torational expectations of therapeutic benefit from metabolically stableTRH-mimetic drugs in a remarkably broad spectrum of clinical situations,both as monotherapy and as an adjunct to other therapeutic agents”.

Narcolepsy with cataplexy is associated with low or undetectable levelsof cerebrospinal fluid (CSF) orexin A levels in about 90% of patients(Baumann and Basset (2005) Sleep Medicine Reviews 9, 253-268). Mutationsof the orexin receptor 2 gene lead to familial canine narcolepsy and aloss of orexin neurons and low CSF orexin A were observed with sporadiccanine narcolepsy. Neurological disorders arising from acute traumaticbrain injury, Guillain-Barre syndrome and advanced Parkinson's syndromemay also be linked with low or undetectable levels of CSF orexin Alevels in some instances. Sakurai has postulated a role for the orexinsystem in feeding and energy homeostasis as the activity of orexinneurons is inhibited by glucose and leptin, and stimulated by ghrelin, astomach-derived peptide which promotes feeding. This may haveimplications for the treatment of obesity (Sakurai (2005) Sleep MedicineReviews 9, 231-241).

The preceding discussion is intended only to facilitate an understandingof the invention. It should not be construed as in any way limiting thescope or application of the following description of the invention, norshould it be construed as an admission that any of the informationdiscussed was within the common general knowledge of the person skilledin the appropriate art at the priority date.

DISCLOSURE OF THE INVENTION

The inventors have discovered that the orexin receptor and thethyrotropin releasing hormone receptor associate. This has importantimplications regarding therapies for ailments associated with eitherreceptor.

Recent studies have shown that GPCRs may not only act as monomers butalso as homo- and hetero-dimers which causes altered ligand binding,signalling and endocytosis (Rios et al. (2000) Pharmacol. Ther. 92,71-87). The effect of drugs acting as agonists or antagonists of aspecific receptor may therefore depend on the binding partners of thisreceptor. It may be desirable to limit the effect of a drug to acellular response mediated by a specific receptor dimer. As Milligan(Milligan, (2006), Drug Discovery Today, 11, 541-549) observes, whilehomo-dimerisation and -oligomerisation have limited implications for thedrug discovery industry, “differential pharmacology, function andregulation of GCPR hetero-dimers and -oligomers suggest means toselectively target GPCRs in different tissues and hint that themechanism of function of several pharmacological agents might bedifferent in vivo than anticipated from simple ligand screeningprogrammes that rely on heterologous expression of a single GPCR”.

The phrase “thyrotropin releasing hormone receptor” or “TRHR” is to beunderstood to at least include the G protein-coupled receptor analogousto that activated by the thyrotropin releasing hormone (TRH) in thethyrotrope cells of the anterior pituitary gland, as well as a number ofstructures in the central nervous system (Riehl et al. (2000)Neuropsychopharmacology 23, 34-45), that has, among other roles, a majorregulatory role in stimulating the synthesis and secretion ofthyrotropin (thyroid-stimulating hormone; TSH) and is synonymous withthyrotropin releasing hormone receptor 1 (TRHR1) (Gershengorn (2003)Thyrotropin-releasing hormone receptor signaling, in Encyclopedia ofhormones. Eds Henry H L and Norman A W. Academic Press. Vol 3; 502-510).The phrase “thyrotropin releasing hormone receptor” or “TRHR” is also tobe understood to mean thyrotropin releasing hormone receptor 2 or TRHR2,a second subtype of thyrotropin releasing hormone receptor known to beexpressed at least in the rat and mouse and whose function is yet to beclearly elucidated (Gershengorn (2003) Thyrotropin-releasing hormonereceptor signaling, in Encyclopedia of hormones. Eds Henry H L andNorman A W. Academic Press. Vol 3; 502-510). The phrase “thyrotropinreleasing hormone receptor” or “TRHR” is to be further understood toinclude newly discovered TRHR family members. Throughout the examples,thyrotropin releasing hormone receptor and the acronym TRHR refers toTRHR1.

The phrase “orexin receptor” or “OxR” is to be understood to mean eitherorexin receptor 1 (OxR1; OXR1; OX₁R; hypocretin-1-receptor; hcrtr 1) ororexin receptor 2 (OxR2; OXR2; OX₂R; hypocretin-2-receptor; hctr 2)being G protein-coupled receptors analogous to those described bySakurai et al. to be activated by orexin A (OxA; hypocretin-1; Hcrt-1)and orexin B (OxB; hypocretin-2; Hcrt-2) (Sakurai et al. (1998) Cell 92,573-585). “Orexin receptor” or “OxR” is to be further understood toinclude newly discovered orexin receptor family members.

In a first aspect of the invention, there is provided a hetero-dimericor hetero-oligomeric receptor, comprising at least one thyrotropinreleasing hormone receptor subunit associated with at least one orexinreceptor subunit.

In a second aspect of the invention, there is provided a method for thetreatment of a patient suffering from an orexin-related ailment byadministering a therapeutically effective amount of athyrotropin-releasing hormone receptor agonist, inverse agonist orantagonist.

In one embodiment, the thyrotropin-releasing hormone receptor agonist,inverse agonist or antagonist may be co-administered with an orexinreceptor agonist, inverse agonist or antagonist.

In a third aspect of the invention, there is provided method for thetreatment of a patient suffering from a thyrotropin-releasinghormone-related ailment by administering a therapeutically effectiveamount of an orexin receptor agonist, inverse agonist or antagonist.

In one embodiment, the orexin receptor agonist, inverse agonist orantagonist may be co-administered with a thyrotropin-releasing hormonereceptor agonist, inverse agonist or antagonist.

In a fourth aspect of the invention, there is provided a method for themanufacture of a medicament for the treatment of a patient sufferingfrom an orexin-related ailment comprising use of a therapeuticallyeffective amount of a thyrotropin releasing hormone receptor agonist,inverse agonist or antagonist.

In one embodiment, the medicament may contain an orexin receptoragonist, inverse agonist or antagonist.

In a fifth aspect of the invention, there is provided a method for themanufacture of a medicament for the treatment of a patient sufferingfrom a thyrotropin-releasing hormone-related ailment comprising use of atherapeutically effective amount of an orexin receptor agonist, inverseagonist or antagonist.

In one embodiment, the medicament may contain a thyrotropin-releasinghormone receptor agonist, inverse agonist or antagonist.

In a sixth aspect of the invention, there is provided a method for thetreatment of a patient suffering from an orexin-related ailment byadministering a therapeutically effective amount of a thyrotropinreleasing hormone-selective binding agent, or fragment thereof.

In one embodiment, the thyrotropin releasing hormone-selective bindingagent is an antibody, including a humanised antibody, a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody and/or an anti-idiotypic antibody.

In a seventh aspect of the invention, there is provided a method for thetreatment of a patient suffering from a thyrotropin-releasinghormone-related ailment by administering a therapeutically effectiveamount of an orexin-selective binding agent, or fragment thereof.

In one embodiment, the orexin-selective binding agent is an antibody,including a humanised antibody, a polyclonal antibody, a monoclonalantibody, a chimeric antibody, a CDR-grafted antibody and/or ananti-idiotypic antibody.

In an eighth aspect of the invention, there is provided a method forscreening a test compound for thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer selective activity, themethod comprising the steps of:

-   -   a) determining whether, and/or the extent to which, the test        compound interacts with the orexin receptor while the orexin        receptor is associated with the thyrotropin releasing hormone        receptor; and    -   b) if the test compound interacts with the orexin receptor while        the orexin receptor is associated with the thyrotropin releasing        hormone receptor, determining whether, or the extent to which        the test compound interacts with the orexin receptor in the        absence of the thyrotropin releasing hormone receptor;        such that a test compound that exhibits greater affinity and/or        potency and/or efficacy when interacting with the orexin        receptor while the orexin receptor is associated with the        thyrotropin releasing hormone receptor is selective for the        thyrotropin releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer.

In a ninth aspect of the invention, there is provided a method forscreening a test compound for thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer selective activity, themethod comprising the steps of:

-   -   a) determining whether, and/or the extent to which, the test        compound interacts with the thyrotropin releasing hormone        receptor while the thyrotropin releasing hormone receptor is        associated with the orexin receptor; and    -   b) if the test compound interacts with the thyrotropin releasing        hormone receptor while the thyrotropin releasing hormone        receptor is associated with the orexin receptor, determining        whether, or the extent to which the test compound interacts with        the thyrotropin releasing hormone receptor in the absence of the        orexin receptor;        such that a test compound that exhibits greater affinity and/or        potency and/or efficacy when interacting with the thyrotropin        releasing hormone receptor while the thyrotropin releasing        hormone receptor is associated with the orexin receptor is        selective for the thyrotropin releasing hormone receptor/orexin        receptor hetero-dimer/-oligomer.

In a tenth aspect of the invention, there is provided a method forscreening a test compound for thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer selective antagonism orpartial agonism, the method comprising the steps of:

-   -   a) determining whether, and/or the extent to which, the test        compound is an antagonist or partial agonist of the thyrotropin        releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer, by contacting said test compound with a        system comprising:        -   i). a first agent, comprising the orexin receptor coupled to            a first reporter component;        -   ii). a second agent, comprising an interacting group coupled            to a second reporter component;        -   iii). a third agent, comprising the thyrotropin releasing            hormone receptor;        -   iv). an agonist of the orexin receptor, the thyrotropin            releasing hormone receptor and/or the thyrotropin releasing            hormone receptor/orexin receptor hetero-dimer/-oligomer;    -    wherein proximity of the first and second reporter components        generates a signal; and wherein the modulator modulates the        association of the interacting group with the thyrotropin        releasing hormone receptor;    -   b) detecting a decrease in the signal as a determination of        whether and/or the extent to which the test compound is an        antagonist or partial agonist of the thyrotropin releasing        hormone receptor/orexin receptor hetero-dimer/-oligomer;    -   c) if the test compound is an antagonist or partial agonist of        the thyrotropin releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer, determining whether, or the extent to        which the test compound is an antagonist or partial agonist of        the thyrotropin releasing hormone receptor in the absence of the        orexin receptor and the orexin receptor in the absence of the        thyrotropin releasing hormone receptor; such that a test        compound that exhibits greater antagonistic or partial agonistic        properties when interacting with the thyrotropin releasing        hormone receptor/orexin receptor hetero-dimer/-oligomer is        selective for the thyrotropin releasing hormone receptor/orexin        receptor hetero-dimer/-oligomer.

In an eleventh aspect of the invention, there is provided a method forscreening a test compound for thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer selective antagonism orpartial agonism, the method comprising the steps of:

-   -   a) determining whether, and/or the extent to which, the test        compound is an antagonist or partial agonist of the thyrotropin        releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer, by contacting said test compound with a        system comprising:        -   i). a first agent, comprising the thyrotropin releasing            hormone receptor coupled to a first reporter component;        -   ii). a second agent, comprising an interacting group coupled            to a second reporter component;        -   iii). a third agent, comprising the orexin receptor;        -   iv). an agonist of the orexin receptor, the thyrotropin            releasing hormone receptor and/or the thyrotropin releasing            hormone receptor/orexin receptor hetero-dimer/-oligomer;    -    wherein proximity of the first and second reporter components        generates a signal; and wherein the modulator modulates the        association of the interacting group with the orexin receptor;        -   b). detecting a decrease in the signal as a determination of            whether and/or the extent to which the test compound is an            antagonist or partial agonist of the thyrotropin releasing            hormone receptor/orexin receptor hetero-dimer/-oligomer;        -   c) if the test compound is an antagonist or partial agonist            of the thyrotropin releasing hormone receptor/orexin            receptor hetero-dimer/-oligomer, determining whether, or the            extent to which the test compound is an antagonist or            partial agonist of the thyrotropin releasing hormone            receptor in the absence of the orexin receptor and the            orexin receptor in the absence of the thyrotropin releasing            hormone receptor; such that a test compound that exhibits            greater antagonistic or partial agonistic properties when            interacting with the thyrotropin releasing hormone            receptor/orexin receptor hetero-dimer/-oligomer is selective            for the thyrotropin releasing hormone receptor/orexin            receptor hetero-dimer/-oligomer.

In a twelfth aspect of the invention, there is provided a method forscreening a test compound for thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer selective inverseagonism, the method comprising the steps of:

-   -   a) determining whether, and/or the extent to which, the test        compound is an inverse agonist of the thyrotropin releasing        hormone receptor/orexin receptor hetero-dimer/-oligomer, by        contacting said test compound with a system comprising:        -   i). a first agent, comprising the orexin receptor coupled to            a first reporter component;        -   ii). a second agent, comprising an interacting group coupled            to a second reporter component;        -   iii). a third agent, comprising a constitutively active            thyrotropin releasing hormone receptor;    -    wherein proximity of the first and second reporter components        generates a signal; and wherein the modulator modulates the        association of the interacting group with the thyrotropin        releasing hormone receptor;    -   b) detecting a decrease in the signal as a determination of        whether and/or the extent to which the test compound is an        inverse agonist of the thyrotropin releasing hormone        receptor/orexin receptor hetero-dimer/-oligomer;    -   c) if the test compound is an inverse agonist of the thyrotropin        releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer, determining whether, or the extent to        which the test compound is an inverse agonist of the thyrotropin        releasing hormone receptor in the absence of the orexin receptor        and the orexin receptor in the absence of the thyrotropin        releasing hormone receptor; such that a test compound that        exhibits greater inverse agonistic properties when interacting        with the thyrotropin releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer is selective for the thyrotropin        releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer.

In a thirteenth aspect of the invention, there is provided a method forscreening a test compound for thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer inverse agonism, themethod comprising the steps of:

-   -   a) determining whether, and/or the extent to which, the test        compound is an inverse agonist of the thyrotropin releasing        hormone receptor/orexin receptor hetero-dimer/-oligomer, by        contacting said test compound with a system comprising:        -   i). a first agent, comprising the thyrotropin-releasing            hormone receptor coupled to a first reporter component;        -   ii). a second agent, comprising an interacting group coupled            to a second reporter component;        -   iii). a third agent, comprising a constitutively active            orexin receptor;    -    wherein proximity of the first and second reporter components        generates a signal; and wherein the modulator modulates the        association of the interacting group with the orexin receptor;    -   b) detecting a decrease in the signal as a determination of        whether and/or the extent to which the test compound is an        inverse agonist of the thyrotropin releasing hormone        receptor/orexin receptor hetero-dimer/-oligomer;    -   c) if the test compound is an inverse agonist of the thyrotropin        releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer, determining whether, or the extent to        which the test compound is an inverse agonist of the thyrotropin        releasing hormone receptor in the absence of the orexin receptor        and the orexin receptor in the absence of the thyrotropin        releasing hormone receptor; such that a test compound that        exhibits greater inverse agonistic properties when interacting        with the thyrotropin releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer is selective for the thyrotropin        releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer.

In the methods of the eighth, ninth, tenth, eleventh, twelfth andthirteenth aspects of the invention, the step of determining whether,and/or the extent to which, the test compound interacts with thethyrotropin releasing hormone receptor while the thyrotropin releasinghormone receptor is associated with the orexin receptor; and/or the stepof determining whether, and/or the extent to which, the test compoundinteracts with the orexin receptor while the orexin receptor isassociated with the thyrotropin releasing hormone receptor may beperformed by way of one or more of the methods described in theapplicant's co-pending international patent application “DetectionSystem and Uses Therefor”, which derives priority from the sameAustralian provisional patent application 2006906292.

In a fourteenth aspect of the invention, there are provided selectiveagonists and/or antagonists and/or inverse agonists of the thyrotropinreleasing hormone receptor/orexin receptor hetero-dimer/-oligomer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 3 are illustrative of the technique by which the associationof thyrotropin releasing hormone receptor and the orexin receptor wasdetected.

FIG. 1 shows the composition of the agents forming the basis of thesystem for detecting molecular associations: A first agent comprises afirst interacting group coupled to a first reporter component; a secondagent comprises a second interacting group coupled to a second reportercomponent; and a third agent comprises a third interacting group.

FIG. 2 shows how the administration of the modulator modulates theassociation of the second interacting group with the third interactinggroup, preferably by interacting with the third interacting group,either alone, or simultaneously with the first interacting group.

FIG. 3 shows that if the first and third interacting groups areassociated, modulation of the association of the second and thirdinteracting groups consequently modulates the proximity of the first andsecond reporter components thereby modulating the signal that is able tobe detected by the detector. Therefore monitoring the signal generatedby proximity of the first and second reporter components by the detectorconstitutes monitoring the association of the first and third agents. Ifthe first and third interacting groups are not associated, the first andsecond reporter components will remain spatially separated andgeneration of a detectable signal is unlikely.

FIG. 4 shows the thyrotropin releasing hormone receptor (TRHR) as IG1,Rluc as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 and a range ofdifferent GPCRs as IG3. eBRET measurements at 37 C were carried out onHEK293 cells transiently co-expressing TRHR/Rluc and barr2/Venus witheither pcDNA3, orexin receptor 2 (OxR2), CXC chemokine receptor 2(CXCR2), hemagglutin epitope-tagged melanocortin receptor 3 or 4(HA-MC3R or HA-MC4R), or dopamine D2 receptor long form (D2LR) or shortform (D2SR) following the treatment of each with their respectiveligands. The different ligand treatment (10⁻⁶M) for each receptor wasthyrotropin releasing hormone (TRH) for TRHR/Rluc (with pcDNA3); orexinA (OxA) for OXR2; interleukin-8 (IL-8) for CXCR2;alpha-melanocyte-stimulating hormone (a-MSH) for HA-MC3R, HA-MC4R; andbromocriptine (BROM) for D2LR and D2SR.

FIG. 5 shows the thyrotropin releasing hormone receptor (TRHR) as IG1,Rluc as RC1, either beta-arrestin 1 (barr1) or beta-arrestin 2 (barr2)as IG2, EGFP as RC2 and OxR2 as IG3. eBRET measurements at 37 C werecarried out on HEK293 cells transiently co-expressing TRHR/Rluc andEGFP/barr1 or EGFP/barr2 with either pcDNA3 or OXR2. Ligand treatmentswere either OxA or TRH only or both OxA and TRH combined.Phosphate-buffered saline (PBS) was used as a vehicle control.

FIG. 6 shows the thyrotropin releasing hormone receptor (TRHR) as IG2,Rluc as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 and OxR1 orOxR2 as IG3. eBRET measurements were carried out at 37 C on HEK293 cellstransiently co-expressing TRHR/Rluc and barr2/Venus with either pcDNA3,OxR1 or OxR2 following pretreatment with 10⁻⁶M OxR1-selectiveantagonist, SB-334867-A, for approximately 40 minutes and then 10⁻⁶M OxA(IG3 ligand; modulator) or 10⁻⁶M TRH (IG1 ligand), or both, was added.Where antagonist was not preincubated, cells were treated with PBSinstead for the same amount of time.

FIG. 7 shows the thyrotropin releasing hormone receptor (TRHR) as IG1,Rluc as RC1, beta-arrestin 1 (barr1) or beta-arrestin 2 (barr2) as IG2,EGFP as RC2 and hemagglutin epitope-tagged OxR2 (HA-OxR2) as IG3. eBRETmeasurements at 37 C were carried out on HEK293 cells transientlyco-expressing TRHR/Rluc and EGFP/barr1 or EGFP/barr2 with either pcDNA3or HA-OxR2. Ligand treatments were either OxA or TRH only.Phosphate-buffered saline (PBS) was used as a vehicle control.

FIG. 8 shows the thyrotropin releasing hormone receptor (TRHR) as IG1,Rluc as RC1, beta-arrestin 1 (barr1)) or beta-arrestin 1phosphorylation-independent mutant R169E (barr1R169E) as IG2, EGFP asRC2 and OxR2 as IG3. eBRET measurements at 37 C were carried out onHEK293 cells transiently co-expressing TRHR/Rluc and EGFP/barr1 orEGFP/barr1R169E with either pcDNA3 or OxR2. Ligand treatments wereeither OxA or TRH only. Phosphate-buffered saline (PBS) was used as avehicle control.

FIG. 9 shows the thyrotropin releasing hormone receptor truncated atamino acid 335 (TRHR335) as IG1, Rluc as RC1, beta-arrestin 1 (barr1) asIG2, EGFP as RC2 and OxR2 or TRHR as IG3. eBRET measurements at 37 Cwere carried out on HEK293 cells transiently co-expressing TRHR335/Rlucand EGFP/barr1 with either OxR2 or TRHR. Ligand treatments were eitherOxA or TRH only.

FIG. 10 shows a dose-response curve for the thyrotropin releasinghormone receptor (TRHR) as IG1, Rluc as RC1, beta-arrestin 2 (barr2) asIG2, Venus as RC2 and in the absence of IG3. BRET measurements at 37 Cwere carried out on HEK293 cells transiently co-expressing TRHR/Rluc,barr2/Venus and pcDNA3 with increasing doses of TRH. Sigmoidal doseresponse curves were plotted using Prism (GraphPad), either assuming aHill slope of 1 or allowing for variable slope. The EC₅₀ and Hill slopevalues for the variable slope curve are included in a table in thegraph.

FIG. 11 shows a dose-response curve for OxR2 as IG1, Rluc as RC1, barr2as IG2, Venus as RC2 and in the absence of IG3. BRET measurements at 37C were carried out on HEK293 cells transiently co-expressing OxR2/Rluc,barr2/Venus and pcDNA3 with increasing doses of OxA. Sigmoidal doseresponse curves were plotted using Prism (GraphPad), either assuming aHill slope of 1 or allowing for variable slope. The EC₅₀ and Hill slopevalues for the variable slope curve are included in a table in thegraph.

FIG. 12 shows dose-response curves for the thyrotropin releasing hormonereceptor (TRHR) as IG1, Rluc as RC1, beta-arrestin 2 (barr2) as IG2,Venus as RC2 and OxR2 as IG3. BRET measurements at 37 C were carried outon HEK293 cells transiently co-expressing TRHR/Rluc, barr2/Venus andOxR2 with increasing doses of OxA. Sigmoidal dose response curves wereplotted using Prism (GraphPad), either assuming a Hill slope of 1 orallowing for variable slope. The EC₅₀ and Hill slope values for thevariable slope curves are included in a table in the graph. Curvesgenerated using coelenterazine h and EnduRen as two forms of Rlucsubstrate (reporter component initiator) are shown.

FIG. 13 shows dose-response curves for TRHR as IG1, Rluc as RC1, barr1as IG2, EGFP as RC2 in the presence or absence of OxR2 as IG3. BRETmeasurements at 37 C were carried out on HEK293 cells transientlyco-expressing TRHR/Rluc and EGFP/barr1 in the absence of OxR2 withincreasing doses of TRH, as well as HEK293 cells transientlyco-expressing TRHR/Rluc and EGFP/barr1 with OxR2 with increasing dosesof OxA with and without 10⁻⁶M TRH. A curve mathematically generated byaddition of the ligand-induced signal generated with 10⁻⁶M TRH (from theTRH: TRHR/Rluc+EGFP/barr1 curve) to each of the points generated for theOxA: TRHR/Rluc+EGFP/barr1+OxR2 curve is also plotted(TRHR/Rluc+EGFP/barr1+OxR2: TRH (10⁻⁶M)+OxA: Data calculated).

FIG. 14 shows dose-response curves for TRHR as IG1, Rluc as RC1, barr1as IG2, EGFP as RC2 in the presence or absence of OxR2 as IG3. BRETmeasurements at 37 C were carried out on HEK293 cells transientlyco-expressing TRHR/Rluc and EGFP/barr1 in the absence of OxR2 withincreasing doses of TRH, as well as HEK293 cells transientlyco-expressing TRHR/Rluc and EGFP/barr1 with OxR2 with increasing dosesof OxA, or increasing doses of TRH with 10⁻⁶M OxA. A curvemathematically generated by addition of the ligand-induced signalgenerated with 10⁻⁶M OxA (from the OxA: TRHR/Rluc+EGFP/barr1+OxR2 curve)to each of the points generated for the TRH: TRHR/Rluc+EGFP/barr1 curveis also plotted (TRHR/Rluc+EGFP/barr1+OxR2: TRH+OxA (10⁻⁶M): Datacalculated).

FIG. 15 shows dose response curves for TRHR335 as IG1, Rluc as RC1,barr2 as IG2, Venus as RC2 and OxR2 as IG3. BRET measurements at 37 Cwere carried out on HEK293 cells transiently co-expressing TRHR335/Rluc,barr2/Venus and OxR2 with increasing doses of TRH and OxA alone or incombination.

FIG. 16 shows cumulative eBRET reads over time for each combination ofreceptors (IG1 and IG3; data captured over 83 mins). TRHR is IG1, Rlucis RC1, barr1 is IG2, EGFP is RC2 and OxR2 is IG3. The same amount ofEGFP/barr1 (IG2-RC2) is transfected for each experiment. TRHR/Rluc(IG1-RC1) is transfected at a constant amount (0.1 μg DNA/well) whileOxR2 (IG3) is transfected at varying amounts of DNA (0, 0.01, 0.05, 0.1,0.5, 0.7 μg DNA/well). eBRET measurements at 37 C were carried out onHEK293 cells following addition of 10⁻⁶M OxA (modulator) to each well.The signal is only detected when OxR2 (IG3) is expressed (no signal wasrecorded at 0 μg OxR2).

FIG. 17 shows dose response curves for TRHR as IG1, Rluc as RC1, barr2as IG2, Venus as RC2 and OxR2 as IG3. BRET measurements at 37 C werecarried out on HEK293 cells transiently co-expressing TRHR/Rluc,barr2/Venus and OxR2 with increasing doses of OxA in either 96-well or384-well microplates.

FIG. 18 shows OxR2 as IG1, Rluc8 as RC1, beta-arrestin 2 (barr2) as IG2,Venus as RC2 and hemagglutin epitope-tagged TRHR (HA-TRHR) as IG3. eBRETmeasurements at 37 C were carried out on HEK293 cells transientlyco-expressing OxR2/Rluc8 and barr2/Venus with either pcDNA3 or HA-TRHR.Ligand treatments were either OxA or TRH only. Phosphate-buffered saline(PBS) was used as a vehicle control. Data presented as ligand-inducedBRET ratios.

FIG. 19 shows the thyrotropin releasing hormone receptor (TRHR) as IG1,Rluc8 as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 andhemagglutin epitope-tagged OxR2 (HA-OxR2) as IG3. eBRET measurements at37 C were carried out on HEK293 cells transiently co-expressingTRHR/Rluc8 and barr2/Venus with HA-OxR2 aliquoted into all wells of a96-well plate. Phosphate-buffered saline (PBS) was added to the firsttwo rows and the last two rows of the 96-well plate (48 wells in total)as a vehicle control. Data presented as fluorescence/luminescence.

FIG. 20 shows the thyrotropin releasing hormone receptor (TRHR) as IG1,Rluc8 as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 andhemagglutin epitope-tagged OxR2 (HA-OxR2) as IG3. eBRET measurements at37 C were carried out on HEK293 cells transiently co-expressingTRHR/Rluc8 and barr2/Venus with HA-OxR2 aliquoted into all wells of a96-well plate. OxA was added to the middle four rows of the 96-wellplate (48 wells in total). Data presented as fluorescence/luminescence.

FIG. 21 shows z-factor data for the thyrotropin releasing hormonereceptor (TRHR) as IG1, Rluc8 as RC1, beta-arrestin 2 (barr2) as IG2,Venus as RC2 and hemagglutin epitope-tagged OxR2 (HA-OxR2) as IG3. Asshown in FIGS. 19 and 20, eBRET measurements at 37 C were carried out onHEK293 cells transiently co-expressing TRHR/Rluc8 and barr2/Venus withHA-OxR2 aliquoted into all wells of a 96-well plate. Phosphate-bufferedsaline (PBS) was added to the first two rows and the last two rows ofthe 96-well plate (48 wells in total) as a vehicle control. OxA wasadded to the middle four rows of the 96-well plate (48 wells in total).Data presented as fluorescence/luminescence.

ABBREVIATIONS

a-MSH alpha-melanocyte-stimulating hormone.

barr beta-arrestin.

BRET Bioluminescence resonance energy transfer.

BROM Bromocriptine.

CB Cannabinoid receptor.

CCR CC chemokine receptor.

CCR5(5)TYFP CCR5 linked to TYFP via a 5 amino acid linker region.

CSF Cerebrospinal fluid.

CXCR CXC chemokine receptor.

D2LR Dopamine D2 receptor (long-form).

D2SR Dopamine D2 receptor (short-form).

DOP Delta opioid.

eBRET extended BRET: BRET monitored over extended time periods.

ECFP Enhanced Cyan Fluorescent Protein, which is a variant of theAequorea victoria green fluorescent protein gene (GFP).

EGFP Enhanced Green Fluorescent Protein is a red-shifted variant ofwild-type GFP.

EYFP Enhanced Yellow Fluorescent Protein.

FRET Fluorescence resonance energy transfer.

GPCRs G-protein coupled receptors.

HA Hemagglutin epitope-tag.

His(6) Histidine tag consisting of 6 consecutive histidine residues.

IG Interacting group.

IL-8 Interleukin-8.

KOP Kappa opioid.

MCP1 Monocyte chemoattractant protein 1 (CCR2 selective ligand).

MCR Melanocortin receptor.

MIP1b Macrophage inflammatory protein 1b (CCR5 selective ligand).

mRFP1 Monomeric red fluorescent protein.

OR Opioid receptor.

OxA Orexin A.

OxB Orexin B.

OxR Orexin receptor.

PBS Phosphate-buffered saline.

pcDNA3 Eukaryotic expression vector.

RC Reporter component.

REM Rapid eye movement.

RET Resonance energy transfer.

Rluc Renilla luciferase.

Rluc8 An improved Renilla luciferase.

SWS Slow wave sleep.

TRH Thyrotropin releasing hormone.

TRHR Thyrotropin releasing hormone receptor.

TYFP Topaz Yellow Fluorescent Protein.

Venus An improved Yellow Fluorescent Protein.

wt Wild type.

BEST MODE(S) FOR CARRYING OUT THE INVENTION

General

All publications, including patents and patent applications, citedherein, whether supra or infra, are hereby incorporated by reference intheir entirety. However, publications mentioned herein are cited for thepurpose of describing and disclosing the protocols, reagents and vectorsthat are reported in the publications and which may be used inconnection with the invention. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

Furthermore, the practice of the present invention employs, unlessotherwise indicated, conventional molecular biology, chemistry andfluorescence techniques, within the skill of the art. Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, eg., Coligan, Dunn, Ploegh, Speicher and Wingfield“Current protocols in Protein Science” (1999) Volume I and II (JohnWiley & Sons Inc.); and Bailey, J. E. and Ollis, D. F., BiochemicalEngineering Fundamentals, McGraw-Hill Book Company, NY, 1986; Lakowicz,J. R. Principles of Fluorescence Spectroscopy, New York : Plenum Press(1983) for fluorescence techniques.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include the plural unless the context clearly dictatesotherwise. Thus, for example, a reference to “a protein” includes aplurality of such proteins, and a reference to “an analyte” is areference to one or more analytes, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any materials andmethods similar or equivalent to those described herein can be used topractice or test the present invention, the preferred materials andmethods are now described.

The invention described herein may include one or more ranges of values(e.g. size, concentration etc). A range of values will be understood toinclude all values within the range, including the values defining therange, and values adjacent to the range that lead to the same orsubstantially the same outcome as the values immediately adjacent tothat value which defines the boundary to the range.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations, such as “comprises” or “comprising”will be understood to imply the inclusion of a stated integer, or groupof integers, but not the exclusion of any other integers or group ofintegers.

Specific

As is apparent from the preceding summary of the invention, theinvention relates, inter alia, to hetero-dimeric or hetero-oligomericreceptor, comprising at least one thyrotropin releasing hormone receptorsubunit associated with at least one orexin receptor subunit. The terms“hetero-dimer” and “hetero-oligomer”, and variations such as“hetero-dimeric” and “hetero-oligomeric”, as used herein, refer to anentity within which at least one thyrotropin releasing hormone receptoris associated with at least one orexin receptor.

The phrase “associated with”, as used herein, refers to combination viaany known direct or indirect stabilising atomic or molecular levelinteraction or any combination thereof, where the interactions include,without limitation, bonding interactions such as covalent bonding, ionicbonding, hydrogen bonding, co-ordinate bonding, or any other molecularbonding interaction, electrostatic interactions, polar or hydrophobicinteractions, or any other classical or quantum mechanical stabilisingatomic or molecular interaction.

Instances of different tissues having different repertoires ofhetero-dimers have been reported. For example, 6′guanidinoaltrindole, ananalogue of a well-known KOP receptor ligand, has been identified as aDOP-KOP hetero-dimer selective agonist, with efficacy as a spinallyselective analgesic, leading to the conclusion that DOP-KOP heterodimersare expressed in the spinal cord, but not in the brain (Waldhoer, M. etal. (2005) A hetero-dimer selective agonist shows in vivo relevance ofG-protein coupled receptor dimers. Proc. Natl. Acad. Sci. USA 102,9050-9055). Accordingly, the hetero-dimeric or hetero-oligomericreceptor, comprising at least one thyrotropin releasing hormone receptorsubunit associated with at least one orexin receptor subunit representsa novel drug target.

As is the case with 6′guanidinoaltrindole, known ligands may exhibitdiffering abilities to trigger a hetero-dimeric receptor, which mayuncover new applications for pre-existing molecules:

-   -   Hilairet et al. 2003 (J. Biol. Chem. 278, 23731-23737) have        recently shown that CB1 antagonists suppress appetite by acting        through a CB1/OxR1 hetero-dimer pair.    -   It has been shown that somatostatin SSTR5 receptor will        hetero-dimerise with a dopamine D2 receptor (Rocheville et        al. (2000) Science 288, 154-157).    -   An angiotensin AT1 receptor/bradykinin B2 receptor hetero-dimer        is believed to be responsible for pre-eclampsia in pregnant        women. Evidence suggests that the hetero-dimer is more sensitive        to Angiotensin II (AbdAlla et al. (2001) Nat. Med. 7,        1003-1009).

As will be apparent from the following examples, the inventors hereinhave identified and characterised the molecular association of thethyrotropin releasing hormone receptor with the orexin receptor.

It will be apparent to a person skilled in the art that association ofthe thyrotropin releasing hormone receptor with orexin receptor enablesthe use of ligands of one receptor (be they agonists, inverse agonistsor antagonists) in the treatment of ailments related to the otherreceptor.

Thus, the present invention encompasses a method for the treatment of apatient suffering from an orexin-related ailment by administering atherapeutically effective amount of a thyrotropin-releasing hormonereceptor agonist, inverse agonist or antagonist.

The thyrotropin-releasing hormone receptor agonist, inverse agonist orantagonist may be co-administered with an orexin receptor agonist,inverse agonist or antagonist.

The present invention further encompasses a method for the treatment ofa patient suffering from a thyrotropin-releasing hormone-related ailmentby administering a therapeutically effective amount of an orexinreceptor agonist, inverse agonist or antagonist.

The present invention further encompasses a method for the manufactureof a medicament for the treatment of a patient suffering from anorexin-related ailment by administering a therapeutically effectiveamount of a thyrotropin releasing hormone receptor agonist, inverseagonist or antagonist.

The medicament may further contain an orexin receptor agonist, inverseagonist or antagonist.

The present invention further encompasses a method for the manufactureof a medicament for the treatment of a patient suffering from athyrotropin-releasing hormone-related ailment by administering atherapeutically effective amount of an orexin receptor agonist, inverseagonist or antagonist.

The medicament may further contain a thyrotropin-releasing hormonereceptor agonist, inverse agonist or antagonist.

Thus, the present invention encompasses a method for the treatment of apatient suffering from an orexin-related ailment by administering atherapeutically effective amount of a thyrotropin-releasinghormone-selective binding agent, or fragment thereof.

The thyrotropin-releasing hormone-selective binding agent may be anantibody, including a humanised antibody, a polyclonal antibody, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/oran anti-idiotypic antibody.

The present invention further encompasses a method for the treatment ofa patient suffering from a thyrotropin-releasing hormone-related ailmentby administering a therapeutically effective amount of anorexin-selective binding agent, or fragment thereof.

The orexin-selective binding agent may be an antibody, including ahumanised antibody, a polyclonal antibody, a monoclonal antibody, achimeric antibody, a CDR-grafted antibody and/or an anti-idiotypicantibody.

The present invention further encompasses a method for the treatment ofa patient suffering from a thyrotropin-releasing hormone-related ailmentor an orexin-related ailment by administering a therapeuticallyeffective amount of a thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer selective agonist, inverse agonist orantagonist.

The present invention further encompasses the use of a therapeuticallyeffective amount of a thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer selective agonist, inverse agonist orantagonist for the manufacture of a medicament for the treatment of apatient suffering from a thyrotropin-releasing hormone-related ailmentor an orexin-related ailment.

The present invention further encompasses a method for the treatment ofa patient suffering from a thyrotropin-releasing hormone-related ailmentby administering a therapeutically effective amount of a selectiveorexin receptor/thyrotropin-releasing hormone receptorhetero-dimer/-oligomer agonist, inverse agonist or antagonist.

The selective orexin receptor/thyrotropin-releasing hormone receptorhetero-dimer/-oligomer agonist, inverse agonist or antagonist may beco-administered with a thyrotropin-releasing hormone receptor agonist,inverse agonist or antagonist.

The selective orexin receptor/thyrotropin-releasing hormone receptorhetero-dimer/-oligomer agonist, inverse agonist or antagonist may beco-administered with an orexin receptor agonist, inverse agonist orantagonist.

The present invention further encompasses a method for the treatment ofa patient suffering from a orexin-related ailment by administering atherapeutically effective amount of a selective orexinreceptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomeragonist, inverse agonist or antagonist.

The selective orexin receptor/thyrotropin-releasing hormone receptorhetero-dimer/-oligomer agonist, inverse agonist or antagonist may beco-administered with a thyrotropin-releasing hormone receptor agonist,inverse agonist or antagonist.

The selective orexin receptor/thyrotropin-releasing hormone receptorhetero-dimer/-oligomer agonist, inverse agonist or antagonist may beco-administered with an orexin receptor agonist, inverse agonist orantagonist.

The present invention further encompasses a method for the manufactureof a medicament for the treatment of a patient suffering from anthyrotropin-releasing hormone-related ailment comprising use of atherapeutically effective amount of a selective orexinreceptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomeragonist, inverse agonist or antagonist.

The medicament may contain an orexin receptor agonist, inverse agonistor antagonist.

The medicament may contain an thyrotropin-releasing hormone receptoragonist, inverse agonist or antagonist.

The present invention further encompasses a method for the manufactureof a medicament for the treatment of a patient suffering from anorexin-related ailment comprising use of a therapeutically effectiveamount of a selective orexin receptor/thyrotropin-releasing hormonereceptor hetero-dimer/-oligomer agonist, inverse agonist or antagonist.

The medicament may contain an orexin receptor agonist, inverse agonistor antagonist.

The medicament may contain an thyrotropin-releasing hormone receptoragonist, inverse agonist or antagonist.

Thyrotropin releasing hormone-related ailments include aliments that arerelated to increased or decreased production of thyrotropin releasinghormone, and/or increased or decreased responsiveness of cells tothyrotropin releasing hormone. The following list (Gary, Keith A., etal., The Thyrotropin-Releasing Hormone (TRH) Hypothesis of HomeostaticRegulation: Implications for TRH-Based Therapeutics, JPET 305:410-416,2003) provides some examples of TRH-related ailments:

-   -   Depression, especially accompanied by hypersomnolence;    -   Chronic fatigue syndromes;    -   Excessive daytime sleepiness (including narcolepsy),        neurasthenia, and lethargy;    -   Sedation secondary to drugs, chemotherapy, or radiation therapy;    -   Sedative intoxication/respiratory distress (ER setting);    -   Recovery from general anesthesia;    -   Attention deficit/hyperactive disorder;    -   Disturbances of circadian rhythm (e.g. jet lag);    -   Bipolar affective disorder as a mood stabilizer*;    -   Anxiety disorders*;    -   Alzheimer's disease and other dementias with cognition        deficits*;    -   Seizure disorders*; and    -   Motor neuron disorders*.        -   *May be particularly effective as adjunctive therapy

However, it should be understood that the phrase thyrotropin releasinghormone-related ailment is not limited thereto.

Orexin-related ailments include aliments that are related to increasedor decreased production of orexin, and/or increased or decreasedresponsiveness of cells to orexin. A major example of an orexin-relatedailment is narcolepsy with cataplexy. This is associated with low orundetectable levels of cerebrospinal fluid (CSF) orexin A levels inabout 90% of patients (Baumann and Bassetti (2005) Sleep MedicineReviews 9, 253-268). Mutations of the orexin receptor 2 gene lead tofamilial canine narcolepsy and a loss of orexin neurons and low CSForexin A were observed with sporadic canine narcolepsy. Neurologicaldisorders arising from acute traumatic brain injury, Guillain-Barresyndrome and advanced Parkinson's syndrome may also be linked with lowor undetectable levels of CSF orexin A levels in some instances. Sakuraihas postulated a role for the orexin system in feeding and energyhomeostasis as the activity of orexin neurons is inhibited by glucoseand leptin, and stimulated by ghrelin, a stomach-derived peptide whichpromotes feeding. This may have implications for the treatment ofobesity (Sakurai (2005) Sleep Medicine Reviews 9, 231-241).

However, it should be understood that the phrase orexin-related ailmentis not limited thereto.

Known orexin receptor modulators include orexin A (OxA; hypocretin-1;Hcrt-1), orexin B (OxB; hypocretin-2; Hcrt-2) and fragments thereof(Lang et al. (2004) J Med Chem 47, 1153-1160).

Known antagonists for both OxR1 and OxR2 include6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogues (Hirose M et al.(2003) Bioorg. Med. Chem. Lett. 13, 4497-4499), Almorexant((2R)-2-{(1S)-6,7-dimethoxy-1-[2-(4-trifluoromethylphenyl)-ethyl}-3,4-dihydro-1H-isoquinolin-2-yl]-N-methyl-2-phenyl-acetamide;ACT-078573; Actelion Pharmaceuticals Ltd., Allschwil, Switzerland;Brisbare-Roch et al. (2007) Nature Medicine 13, 150-155).

Known OxR1 antagonists include SB-334867-A(1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl ureahydrochloride), SB-674042(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2ylmethyl)-pyrrolidin-1-yl)-methanone),SB-408124(1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea)and SB-410220(1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) (Hayneset al. (2000) Regulatory Peptides 96, 45-51; Langmead et al. (2004)British Journal of Pharmacology 141, 340-346).

Known OxR2 antagonists include N-Arylmethyl tert-leucyl6,7-dimethoxy-1,2,3,4-tetrahydroiso-quinoline analogues and N-acyl6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogues (Hirose M et al.(2003) Bioorg. Med. Chem. Lett. 13, 4497-4499), and substituted4-phenyl-[1,3]dioxanes, particularly1-(2,4-dibromo-phenyl)-3-((4S,5S)-2,2-dimethyl-4-phenyl-[1,3]dioxan-5-yl)-urea(McAtee L C et al. (2004) Bioorg. Med. Chem. Lett. 14, 4225-4229).

Known modulators of the thyrotropin releasing hormone receptor includethyrotropin releasing hormone (TRH; thyroliberin; TRF;pGlu-His-Pro-NH₂), [Glu2]TRH, [Glu2]TRH with the amino-terminalpyroglutamyl residue replaced with a pyridinium moiety (Prokai-Tatrai etal. (2005) Med. Chem. 1, 141-152), methyl-TRH, (3-methyl-His2)TRH,montirelin ((3R,6R)-6-methyl-5-oxo-3-thiomorpholinylcarbonyl-L-histidyl-L-prolinamide tetrahydrate; CG-3703; GrunenthalGmbH, Aachen, Germany), CNK-602A (N-[(6-methyl-5-oxo-3-thiomorpholinyl)carbonyl]-L-histidyl-L-prolinamide; Renming et al. (1992) Eur. J.Pharmacol. 223, 185-192), taltirelin((−)-N-RS)-hexahydro-1-methyl-2,6-dioxo-4-pyrimidinylcarbonyl]-L-histidyl-L-prolinamidetetrahydrate; Ceredist; TA-0910; Tanabe Seiyaku Co., Ltd., Osaka,Japan), JTP-2942(N^(alpha)-[(1S,2R)-2-methyl-4-oxocyclopentylcarbonyl]-L-histidyl-L-prolinamidemonohydrate; Japan Tobacco, Inc., Tokyo, Japan), azetirelin (YM-14673;Yamanouchi Pharmaceutical Co., Ltd, Tokyo, Japan), DN-1417(Gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate;Miyamoto M et al. (1981) Life Sci. 28, 861-869), RX-77368(pGlu-His-(3,3′-dimethyl)-Pro-NH₂; Ferring Pharmaceuticals, Feltham,Middlesex, UK), CG-3509 (Grunenthal GmBH, Stolberg, Germany), MK-771(1-pyro-2-aminoadipyl-L-histidyl-L-thiazolidine-4-carboxamide; Merck,Rahway, N.J.), posatirelin (RGH 2202;L-6-ketopiperidine-2-carbonyl-L-leucyl-L-proline amide; Gedeon RichterPharmaceuticals, Budapest, Hungary), Ro 24-9975(1S,3R,5(2S),5S)-5-[(5-oxo-1-phenylmethyl)-2-pyrrolidinyl]-methyl]-5-[(1H-imidazol-5-yl)methyl]-cyclohexaneacetamide;Hoffman-La Roche, Basel, Switzerland), protirelin(5-oxo-L-prolyl-L-histidyl-L-proline amide; Thyrel® TRH; FerringPharmaceuticals, Tarrytown, N.Y.), midazolam, diazepam andchlordiazepoxide (inverse agonists; Jinsi-Parimoo A and Gershengorn MC(1997) Endocrinology 138, 1471-1475).

A strong association between the orexin system and narcolepsy withcataplexy has been established (Sakurai (2005) Sleep Medicine Reviews 9,231-241). Furthermore, Nishino et al. suggest that TRH analogs may beuseful for the treatment of excessive daytime sleepiness in narcolepsy(Nishino et al. (1997) The Journal of Neuroscience 17, 6401-6408). TheTRH analogs CG-3703 and TA-0910 significantly reduced slow wave sleep(SWS) and rapid eye movement (REM) sleep in a dose- and time-dependentmanner. Furthermore, the TRH analogs completely suppressed cataplexy inmost of the animals studied. Serum T₃ and T₄ did not changesignificantly “suggesting that the anticataplectic and alerting effectsof TRH and analogs of TRH are mediated by neuromodulatory CNS propertiesand not by indirect effects on the thyroid axis.” (Nishino et al. (1997)The journal of neuroscience 17, 6401-6408). These observations weresupported by a further study in 2000 (Riehl et al. (2000)Neuropsychopharmacology 23, 34-45). The mode of action of TRH andorexins (and analogs thereof) in the pathophysiology of narcolepsyremains to be elucidated, however, the hetero-dimer/-oligomerinteraction identified in this invention contributes to the integrationof these receptor systems. Riehl et al. comment, “The mechanismunderlying the involvement of the hypocretin system in thepathophysiology of narcolepsy remains unclear. It is interesting tonote, however, that hypocretin [orexin]-containing neurons areexclusively localized in the lateral hypothalamus (Sakurai et al. 1998[Cell, 92, 573-585]; Peyron et al. 1998 [J. Neurosc. 18, 9996-10015]),an area that is rich in TRH neurons (Kreider et al. 1985 [Peptides 6,997-1000]). In addition, both hypocretin [orexin] and TRH receptors areG-protein coupled receptors for neuropeptides, and that the TRH receptorexhibits the second highest (25%) homology (with the Y2 neuropeptide Yreceptor having the highest homology) to the hypocretin [orexin]receptors (Sakurai et al. 1998 [Cell, 92, 573 -585]), suggesting thatTRH may play an important role in the pathophysiology of narcolepsythrough an unknown specific interaction with the hypocretin [orexin]system.” (Riehl et al. (2000) Neuropsychopharmacology 23, 34-45). Theauthors have identified the likelihood of TRH and orexin systemintegration without identifying that such integration could occur as aresult of the receptor hetero-dimerization/-oligomerization identifiedin this invention.

In addition to narcolepsy, the TRH and orexin receptor systems mayintegrate with regard to the control of feeding and metabolichomeostasis. Thyroid hormone secretion is suppressed during starvation,whereas preprohypocretin (the precursor of orexin peptides) mRNA isupregulated in the lateral hypothalamus. Such observations led Kok atal. to investigate the integration of the TRH and orexin systems as,“although the topography of hypocretin- [orexin-] and thyrotrope neuralcircuits suggests that TRH neuronal activity is governed by hypocretin[orexin] input, the nature of the signal (i.e. excitatory or inhibitory)remains unclear” (Kok at al. (2005) AJP—Endocrinology and Metabolism288, 892-899). This study demonstrated significantly lower averageplasma TSH concentrations in orexin-deficient narcoleptic humanscompared to controls. It is important to note that, as well asfeedforward signalling, complex feedback pathways involving autocrineand paracrine feedback via receptors expressed on or in the locality ofhormone-/neurotransmitter-secreting neurons are likely to be common insuch systems and may play a physiological or pathophysiological role insystem integration where these receptors form hetero-dimers/-oligomers.

In one embodiment, the present invention provides a method for thetreatment of a patient suffering from an orexin-related ailment otherthan narcolepsy by administering a therapeutically effective amount of athyrotropin-releasing hormone receptor agonist, inverse agonist orantagonist.

In one embodiment, the present invention provides a method for thetreatment of a patient suffering from an orexin-related ailment byadministering a therapeutically effective amount of athyrotropin-releasing hormone receptor agonist, inverse agonist orantagonist other than TA0910 (Ceredist).

In one embodiment, the present invention provides a method for thetreatment of a patient suffering from an orexin-related ailment byadministering a therapeutically effective amount of athyrotropin-releasing hormone receptor agonist, inverse agonist orantagonist other than TA0910 (Ceredist), CG3703 and CG3509.

In one embodiment, the present invention provides a method for thetreatment of a patient suffering from an orexin-related ailment byadministering a therapeutically effective amount of athyrotropin-releasing hormone receptor agonist, inverse agonist orantagonist selected from the group: thyrotropin releasing hormone (TRH;thyroliberin; TRF; pGlu-His-Pro-NH₂), [Glu2]TRH, [Glu2]TRH with theamino-terminal pyroglutamyl residue replaced with a pyridinium moiety(Prokai-Tatrai et al. (2005) Med. Chem. 1, 141-152), methyl-TRH,(3-methyl-His2)TRH, montirelin ((3R,6R)-6-methyl-5-oxo-3-thiomorpholinylcarbonyl-L-histidyl-L-prolinamide tetrahydrate; CG-3703; GrunenthalGmbH, Aachen, Germany), CNK-602A (N-[(6-methyl-5-oxo-3-thiomorpholinyl)carbonyl]-L-histidyl-L-prolinamide; Renming et al. (1992) Eur. J.Pharmacol. 223, 185-192), JTP-2942(N^(alpha)-[(1S,2R)-2-methyl-4-oxocyclopentylcarbonyl]-L-histidyl-L-prolinamidemonohydrate; Japan Tobacco, Inc., Tokyo, Japan), azetirelin (YM-14673;Yamanouchi Pharmaceutical Co., Ltd, Tokyo, Japan), DN-1417(Gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate;Miyamoto M et al. (1981) Life Sci. 28, 861-869), RX-77368(pGlu-His-(3,3′-dimethyl)-Pro-NH₂; Ferring Pharmaceuticals, Feltham,Middlesex, UK), CG-3509 (Grunenthal GmBH, Stolberg, Germany), MK-771(1-pyro-2-aminoadipyl-L-histidyl-L-thiazolidine-4-carboxamide; Merck,Rahway, N.J.), posatirelin (RGH 2202;L-6-ketopiperidine-2-carbonyl-L-leucyl-L-proline amide; Gedeon RichterPharmaceuticals, Budapest, Hungary), Ro 24-9975(1S,3R,5(2S),5S)-5-[(5-oxo-1-phenylmethyl)-2-pyrrolidinyl]-methyl]-5-[(1H-imidazol-5-yl)methyl]-cyclohexaneacetamide;Hoffman-La Roche, Basel, Switzerland), protirelin(5-oxo-L-prolyl-L-histidyl-L-proline amide; Thyrel® TRH; FerringPharmaceuticals, Tarrytown, N.Y.), midazolam, diazepam andchlordiazepoxide (inverse agonists; Jinsi-Parimoo A and Gershengorn M C(1997) Endocrinology 138, 1471-1475).

In one embodiment, the present invention provides a method for thetreatment of a patient suffering from an orexin-related ailment byadministering a therapeutically effective amount of athyrotropin-releasing hormone receptor agonist, inverse agonist orantagonist selected from the group: thyrotropin releasing hormone (TRH;thyroliberin; TRF; pGlu-His-Pro-NH₂), [Glu2]TRH, [Glu2]TRH with theamino-terminal pyroglutamyl residue replaced with a pyridinium moiety(Prokai-Tatrai et al. (2005) Med. Chem. 1, 141-152), methyl-TRH,(3-methyl-His2)TRH, CNK-602A (N-[(6-methyl-5-oxo-3-thiomorpholinyl)carbonyl]-L-histidyl-L-prolinamide; Renming et al. (1992) Eur. J.Pharmacol. 223, 185-192), JTP-2942(N^(alpha)-[(1S,2R)-2-methyl-4-oxocyclopentylcarbonyl]-L-histidyl-L-prolinamidemonohydrate; Japan Tobacco, Inc., Tokyo, Japan), azetirelin (YM-14673;Yamanouchi Pharmaceutical Co., Ltd, Tokyo, Japan), DN-1417(Gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate;Miyamoto M et al. (1981) Life Sci. 28, 861-869), RX-77368(pGlu-His-(3,3′-dimethyl)-Pro-NH₂; Ferring Pharmaceuticals, Feltham,Middlesex, UK), MK-771(1-pyro-2-aminoadipyl-L-histidyl-L-thiazolidine-4-carboxamide; Merck,Rahway, N.J.), posatirelin (RGH 2202;L-6-ketopiperidine-2-carbonyl-L-leucyl-L-proline amide; Gedeon RichterPharmaceuticals, Budapest, Hungary), Ro 24-9975(1S,3R,5(2S),5S)-5-[(5-oxo-1-phenylmethyl)-2-pyrrolidinyl]-methyl]-5-[(1H-imidazol-5-yl)methyl]-cyclohexaneacetamide;Hoffman-La Roche, Basel, Switzerland), protirelin(5-oxo-L-prolyl-L-histidyl-L-proline amide; Thyrel® TRH; FerringPharmaceuticals, Tarrytown, N.Y.), midazolam, diazepam andchlordiazepoxide (inverse agonists; Jinsi-Parimoo A and Gershengorn M C(1997) Endocrinology 138, 1471-1475).

The present invention comprises a method for screening a test compoundfor thyrotropin releasing hormone receptor/orexin receptorhetero-dimer/-oligomer selective activity, the method comprising thesteps of:

-   -   a) determining whether, and/or the extent to which, the test        compound interacts with the orexin receptor while the orexin        receptor is associated with the thyrotropin releasing hormone        receptor; and    -   b) if the test compound interacts with the orexin receptor while        the orexin receptor is associated with the thyrotropin releasing        hormone receptor, determining whether, or the extent to which        the test compound interacts with the orexin receptor in the        absence of the thyrotropin releasing hormone receptor;        such that a test compound that exhibits greater affinity and/or        potency and/or efficacy when interacting with the orexin        receptor while the orexin receptor is associated with the        thyrotropin releasing hormone receptor is selective for the        thyrotropin releasing hormone receptor/orexin receptor        hetero-dimer/-oligomer.

The present invention comprises a method for screening a test compoundfor thyrotropin releasing hormone receptor/orexin receptorhetero-dimer/-oligomer selective activity, the method comprising thesteps of:

-   -   a) determining whether, and/or the extent to which, the test        compound interacts with the thyrotropin releasing hormone        receptor while the thyrotropin releasing hormone receptor is        associated with the orexin receptor; and    -   b) if the test compound interacts with the thyrotropin releasing        hormone receptor while the thyrotropin releasing hormone        receptor is associated with the orexin receptor, determining        whether, or the extent to which the test compound interacts with        the thyrotropin releasing hormone receptor in the absence of the        orexin receptor;        such that a test compound that exhibits greater affinity and/or        potency and/or efficacy when interacting with the thyrotropin        releasing hormone receptor while the thyrotropin releasing        hormone receptor is associated with the orexin receptor is        selective for the thyrotropin releasing hormone receptor/orexin        receptor hetero-dimer/-oligomer.

In a preferred embodiment of the invention, the step of determiningwhether, and/or the extent to which, the test compound interacts withthe thyrotropin releasing hormone receptor while the thyrotropinreleasing hormone receptor is associated with the orexin receptor;and/or the step of determining whether, and/or the extent to which, thetest compound interacts with the orexin receptor while the orexinreceptor is associated with the thyrotropin releasing hormone receptorare performed by way of the methods described in the applicant'sco-pending international patent application “Detection System and UsesTherefor”, which derives priority from the same Australian provisionalpatent application 2006906292.

The present invention includes selective agonists and/or antagonistsand/or inverse agonists of the thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer.

As used herein the term “patient” refers to any animal that may besuffering from one or more of orexin- or thyrotropin releasinghormone-related ailments. Most preferably the animal is a mammal. Theterm will be understood to include for example human, farm animals(i.e., cattle, horses, goats, sheep and pigs), household pets (i.e.,cats and dogs) and the like.

The phrase “therapeutically effective amount” as used herein refers toan amount sufficient to modulate a biological activity associated withthe interaction of orexin receptor agonist, inverse agonist orantagonist with the orexin receptor or thyrotropin releasing hormonereceptor agonist, inverse agonst or antagonist with thethyrotropin-releasing hormone receptor or of orexinreceptor/thyrotropin-releasing hormone receptorhetero-dimer/oligomer-specific agonist, inverse agonist or antagonistwith an orexin receptor/thyrotropin-releasing hormone receptorhetero-dimer/oligomer. In the context of aspects of the invention whereboth a thyrotropin-releasing hormone receptor agonist, inverse agonistor antagonist and a orexin receptor agonist, inverse agonist orantagonist are administered in combination, a therapeutically effectiveamount of a thyrotropin-releasing hormone receptor agonist, inverseagonist or antagonist or a therapeutically effective amount of an orexinreceptor agonist, inverse agonist or antagonist in combination may belower than therapeutically effective amounts of thyrotropin-releasinghormone receptor agonist, inverse agonist or antagonist or orexinreceptor agonist, inverse agonist or antagonist when administered alone.That is, the administration of a thyrotropin-releasing hormone receptoragonist, inverse agonist or antagonist and a orexin receptor agonist,inverse agonist or antagonist in combination may generate a therapeuticeffect at what would otherwise be sub-therapeutic doses of either.

Medicaments of the invention may be administered by injection, orprepared for oral, pulmonary, nasal or for any other form ofadministration. Preferably the medicaments are administered, forexample, intravenously, subcutaneously, intramuscularly, intraorbitally,ophthalmically, intraventricularly, intracranially, intracapsularly,intraspinally, intracisternally, intraperitoneally, buccal, rectally,vaginally, intranasally or by aerosol administration.

The mode of administration must, however, be at least suitable for theform in which the medicament has been prepared. The mode ofadministration for the most effective response may need to be determinedempirically and the means of administration described below are given asexamples, and do not limit the method of delivery of the composition ofthe present invention in any way. All the above formulations arecommonly used in the pharmaceutical industry and are commonly known tosuitably qualified practitioners.

In addition to the agonist(s) and/or inverse agonist(s) and/orantagonist(s), the medicaments of the invention may includepharmaceutically acceptable nontoxic excipients and carriers andadministered by any parenteral techniques such as subcutaneous,intravenous and intraperitoneal injections. In addition the formulationsmay optionally contain one or more adjuvants.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water-soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. Alternatively, the compounds of the inventionmay be encapsulated in liposomes and delivered in injectable solutionsto assist their transport across cell membrane. Alternatively or inaddition such preparations may contain constituents of self-assemblingpore structures to facilitate transport across the cellular membrane.The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propylene glycoland liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. Proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in the required amount in an appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the various sterilised active ingredient into a sterilevehicle that contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying techniquesthat yield a powder of the active ingredient plus any additional desiredingredient from previously sterile-filtered solution thereof.

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Martin, Remington's Pharmaceutical Sciences, 18thEd. (1990 Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatised with various polymers (E.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, in Modern Pharmaceutics, Chapter 10,Banker and Rhodes ed., (1979), herein incorporated by reference. Ingeneral, the formulation will include the compounds described as part ofthe invention (or a chemically modified form thereof), and inertingredients which allow for protection against the stomach environment,and release of the biologically active material in the intestine.

For the agonists, antagonists and inverse agonists of the invention thelocation of release may be the stomach, the small intestine (theduodenum, the jejunum, or the ileum), or the large intestine. Oneskilled in the art has available formulations that will not dissolve inthe stomach, yet will release the material in the duodenum or elsewherein the intestine. Preferably, the release will avoid the deleteriouseffects of the stomach environment, either by protection of thecomposition or by release of the compounds beyond the stomachenvironment, such as in the intestine.

To ensure full gastric resistance, a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings that make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, moulded tablets or tablet triturates,moist massing techniques can be used.

The therapeutic can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colourants and flavouring agents may all be included. For example,compounds may be formulated (such as by liposome or microsphereencapsulation) and then further contained within an edible product, suchas a refrigerated beverage containing colorants and flavouring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, alpha-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrants include but are notlimited to starch including the commercial disintegrant based on starch,Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic compounds together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methylcellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to: stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, and Carbowax4000 and 6000.

Glidants that might improve the flow properties of the compound duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment, asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the compounds eitheralone or as a mixture in different ratios.

Additives which potentially enhance uptake of the compounds are forinstance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The compounds could beincorporated into an inert matrix that permits release by eitherdiffusion or leaching mechanisms i.e., gums. Slowly degeneratingmatrices may also be incorporated into the formulation. Another form ofa controlled release of this therapeutic is by a method based on theOros therapeutic system (Alza Corp.), i.e. the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidized bed orby compression coating.

Also contemplated herein is pulmonary delivery of the compounds. Thecompounds may be delivered to the lungs of a mammal while inhaling andtraverses across the lung epithelial lining to the blood stream.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered-doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the compounds. Typically, each formulation is specific tothe type of device employed and may involve the use of an appropriatepropellant material, in addition to the usual diluents, adjuvants and/orcarriers useful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the compounds suspended in water.The formulation may also include a buffer and a simple sugar (e.g., forprotein stabilization and regulation of osmotic pressure). The nebulizerformulation may also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the compounds caused by atomization of thesolution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the compounds suspended in apropellant with the aid of a surfactant. The propellant may be anyconventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the compound and may also include abulking agent, such as lactose, sorbitol, sucrose, or mannitol inamounts which facilitate dispersal of the powder from the device, e.g.,50 to 90% by weight of the formulation. The compounds (or derivative)should most advantageously be prepared in particulate form with anaverage particle size of less than 10 microns, most preferably 0.5 to 5microns, for most effective delivery to the distal lung.

Nasal delivery of the compounds is also contemplated. Nasal deliveryallows the passage of the protein to the blood stream directly afteradministering the therapeutic product to the nose, without the necessityfor deposition of the product in the lung. Formulations for nasaldelivery include those with dextran or cyclodextran.

It will be appreciated that the medicaments of the invention may begiven as a single dose schedule, or preferably, in a multiple doseschedule. A multiple dose schedule is one in which a primary course ofdelivery may be with 1 to 10 separate doses, followed by other dosesgiven at subsequent time intervals required to maintain or reinforce thetreatment. The dosage regimen will also, at least in part, be determinedby the need of the individual and the judgement of the practitioner.

The invention will now be further described by way of reference only tothe following non-limiting examples. It should be understood, however,that the examples following are illustrative only, and should not betaken in any way as a restriction on the generality of the inventiondescribed above.

EXAMPLES General Methods

Briefly, referring to FIGS. 1 to 3, the IGs are provided in the form ofthe two receptors (TRHR and OxR). One of the two is attached to an RC(IG1-RC1, IG3). A second IG (IG2-RC2) is derived from a molecule thatinteracts with the receptors upon ligand binding (e.g. beta-arrestin, ora mutant thereof). The detection system not only detects the formationof the TRHR-OxR heterodimer but can distinguish whether a ligand or drugacts as an agonist, partial agonist, antagonist, inverse agonist orpartial inverse agonist at the receptor hetero-dimer.

Cells were seeded in 6-well plates at a density of approximately 630,000cells/well (HEK293FT) or approximately 150,000 cells/well (COS-7) andmaintained at 37° C., 5% CO2 in Complete Media (DMEM containing 0.3mg/ml glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin(Gibco)) supplemented with 10% fetal calf serum (FCS; Gibco). Transienttransfections were carried out 24 h after seeding using GeneJuice(Novagen) or Metafectene (Biontex) according to manufacturerinstructions. 24 h post-transfection, cells were washed with PBS,detached using 0.05% trypsin/0.53 mM EDTA, resuspended in HEPES-bufferedphenol red free Complete Media containing 5% FCS and added to apoly-L-lysine-coated white microplate (Nunc). 48 h post-transfection,eBRET assays were carried out following pre-incubation of cells withEnduRen™ (Promega) at a final concentration of 30-40 μM, at 37° C., 5%CO₂ for 2 h. For original BRET studies, the HEPES-buffered phenol redfree Complete Media was replaced with PBS and coelenterazine h(Molecular Probes) added to a final concentration of 5 μM immediatelyprior to commencing the assay. BRET measurements were taken at 37° C.using the Victor Light plate reader with Wallac 1420 software(Perkin-Elmer). Filtered light emissions were sequentially measured for3-5 s in each of the ‘donor wavelength window’ (400-475 nm) and‘acceptor wavelength window’ (>500 nm for EGFP or 520-540 nm for EYFP,Topaz (TYFP) or Venus). The BRET signal observed between interactingproteins is normalized by subtracting the background BRET ratio. Thiscan be done in one of two ways (see Pfleger et al. (2006) Cell Signal18, 1664-1670; Pfleger et al. (2006) Nat Protoc 1, 336-344): 1) theratio of light emission through the acceptor wavelength window over the400-475 nm emission for a cell sample containing only the donorconstruct is subtracted from the same ratio for a sample containing theinteracting acceptor and donor fusion proteins; 2) the ratio of lightemission through the ‘acceptor wavelength window’ over the 400-475 nmemission for a cell sample treated with vehicle is subtracted from thesame ratio for a second aliquot of the same cell sample treated withligand. In the following examples, the first calculation will be used,unless the signal is described as the ‘ligand-induced BRET ratio’.Alternatively, and particularly when illustrating z-factor data (Zhanget al. (1999) J Biomol Screen 4, 67-73), the BRET signal observedbetween interacting proteins can be shown in conjunction with (as opposeto being subtracted by) the background BRET ratio to evaluate errorassociated with the BRET signal observed between interacting proteinsand the error associated with the background BRET ratio independently.In this case, data are shown as ‘fluorescence/luminescence’ being theratio of light emission through the ‘acceptor wavelength window’ overthe 400-475 nm emission for a particular cell sample. Unless otherwisestated, BRET signals were measured in 96-well microplates.

Example 1 Measurement of a Detectable Signal Indicative of the MolecularAssociation of the Thyrotropin Releasing Hormone Receptor with theOrexin Receptor

Referring now to FIG. 4, eBRET signals were measured from cellstransiently co-expressing TRHR/Rluc and barr2/Venus with either pcDNA3,OxR2, CXCR2, HA-MC3R, HA-MC4R, D2LR or D2SR following the treatment ofeach with their respective ligands.

Prior to ligand treatment (added at 0 minutes), a baseline eBRET signalwas recorded for each of the receptor combinations. Within the firstminute, TRH treatment of cells co-expressing TRHR/Rluc and barr2/Venuswith pcDNA3, resulted in the eBRET signal rapidly reaching a peak ofgreater than 0.17 and this signal remained high for the entire recordingperiod (nearly 2 hours). A signal was also observed following OxAtreatment of cells co-expressing TRHR/Rluc, barr2/Venus and OxR2. Thissignal however took up to 30 minutes to reach approximately 0.07-0.08.No ligand-induced eBRET signals were observed for any of the otherreceptor combinations.

This example demonstrates that a signal resulting from the proximity ofRC1 and RC2 is detected specifically for the combination where thethyrotropin releasing hormone receptor (TRHR) is IG1, Rluc is RC1,beta-arrestin 2 (barr2) is IG2, Venus is RC2 and OxR2 is IG3, and whenthe modulator, in this case OxA, modulates the association of IG2 andIG3 as a result of interacting specifically with IG3. A signal is notdetected when IG3 is CXCR2, HA-MC3R, HA-MC4R, D2LR or D2SR and agonistsspecific for these IG3s modulate the association of IG2 and IG3,demonstrating the specificity of the signal for the combination withOxR2 as IG3.

This example demonstrates that the inventors have identified themolecular association of the thyrotropin releasing hormone receptor withthe orexin receptor.

This example further demonstrates that the kinetic profile observed forthe signal resulting from RC1 and RC2 proximity due to modulation of theassociation of IG2 and IG3 is distinct from the kinetic profile observedfor an eBRET signal resulting from RC1 and RC2 proximity due toassociation of IG1 and IG2 when this IG1-IG2 association is modulated byligand, in this case TRH, interacting specifically with IG1. The formerprofile is substantially delayed compared to the latter profile.

Example 2 Measurement of Additive Detectable Signals Indicative of theMolecular Association of the Thyrotropin Releasing Hormone Receptor withthe Orexin Receptor

Referring now to FIG. 5, eBRET signals were measured from cellstransiently co-expressing TRHR/Rluc and EGFP/barr1 or EGFP/barr2 witheither pcDNA3 or OXR2. Ligand treatments were either OxA or TRH only orboth OxA and TRH combined.

Prior to ligand treatment (added at 0 minutes), baseline eBRET signalswere recorded for each of the receptor combinations. PBS treated cellsexpressing each of the combinations exhibited only baseline eBRETsignals for the entire recording period (70 minutes). Followingtreatment with OxA, cells expressing OxR2 and either EGFP/barr1(crosses) or EGFP/barr2 (grey inverted triangles) exhibited an eBRETsignal reaching a plateau after more than 10 minutes. In cellsexpressing TRHR/Rluc only (no OxR2) with either of the beta arrestins,TRH treatment resulted in a rapid stimulation of the eBRET signal. Thesignal with barr2 (black circles) was greater than that for barr1 (greytriangles) however there was no difference for either beta arrestin ifOxA was present (barr2, black triangles; barr1, grey circles). In cellsexpressing both TRHR/Rluc and OxR2 (barr1, grey squares; barr2, blacksquares), the addition of both ligands showed an increased eBRET signalover and above that seen following addition of OxA or TRH alone.

This example demonstrates that a signal resulting from the proximity ofRC1 and RC2 is detected for the combination where the thyrotropinreleasing hormone receptor (TRHR) is IG1, Rluc is RC1, eitherbeta-arrestin 1 (barr1) or beta-arrestin 2 (barr2) is IG2, EGFP is RC2and OxR2 is IG3 when the modulator, OxA, modulates the association ofIG2 and IG3 as a result of interacting specifically with IG3.

Therefore, this example demonstrates signal detection using analternative combination from that shown in example 1, including use of adifferent IG2 and RC2.

As in example 1, this example demonstrates the delayed kinetic profileobserved for the signal resulting from RC1 and RC2 proximity due tomodulation of the association of IG2 and IG3, in this case by OxA, asdistinct from the more rapid kinetic profile observed for an eBRETsignal resulting from RC1 and RC2 proximity due to association of IG1and IG2 when this IG1-IG2 association is modulated by ligand, in thiscase TRH, interacting specifically with IG1. However, in addition tothat demonstrated in example 1, this example demonstrates the additiveeffect of combined treatment with IG1 ligand (TRH) and IG3 ligand (OxA;modulator).

Therefore, this example provides further and distinct evidence for themolecular association of the thyrotropin releasing hormone receptor withthe orexin receptor, as this additive effect is indicative of RC1 andRC2 proximity as a result of IG1-IG2 association in addition toIG2-IG3-IG1 association. This provides evidence against signalsoriginating from non-specific IG1-IG2 association in the absence of anIG1-specific ligand. Without wishing to be bound by theory, thisadditive effect may also be partly due to IG1 ligand acting as amodulator to modulate the association of IG2 and IG3 via allostericeffects on IG3. Furthermore, this additive effect may also be partly dueto an active IG conformation (one that is bound to agonist) being morefavourable for signal generation, perhaps enabling increased proximityof RC1 and RC2, or more favourable relative orientation of RC1 and RC2.

Example 3 Measurement of the Effect on Signal Generation of anAntagonist that Competes for Modulator Binding

Referring now to FIG. 6, eBRET signals were measured from cellstransiently co-expressing TRHR/Rluc and barr2/Venus with either pcDNA3,OxR1 or OxR2 following pretreatment with 10⁻⁶M OxR1-selectiveantagonist, SB-334867-A, for approximately 40 minutes prior to additionof 10⁻⁶M OxA (IG3 ligand; modulator) or 10⁻⁶M TRH (IG1 ligand), or both.Cells not pretreated with antagonist were pretreated with PBS insteadfor the same amount of time.

Prior to agonist treatment (added at 0 minutes), baseline eBRET signalwas recorded for each of the receptor combinations. A small eBRET signalwas observed for OxA-treated TRHR/Rluc and barr2/Venus and OxR1 (greydiamonds). This signal was reduced in the presence of antagonist (opensquares). The addition of both TRH and OxA to the OxR1-expressing cellsresulted in a much larger signal (white triangles) and the size of thissignal was reduced in the presence of the antagonist (grey circles). AneBRET signal was observed following OxA treatment of cells co-expressingTRHR/Rluc, barr2/Venus and OxR2 (black diamonds). This signal was notaffected by the pre-treatment of antagonist (white squares). Theaddition of both TRH and OxA to the OxR2-expressing cells resulted in asignal that did not differ in either the presence (black circles) or theabsence (grey triangles) of antagonist.

This example shows a signal resulting from the proximity of RC1 and RC2detected for the combination where the thyrotropin releasing hormonereceptor (TRHR) is IG1, Rluc is RC1, beta-arrestin 2 (barr2) is IG2,Venus is RC2 and OxR1 or OxR2 is IG3 when the modulator, OxA, modulatesthe association of IG2 and IG3 as a result of interacting specificallywith IG3.

This example demonstrates that specific antagonism of modulator binding,in this case the specific antagonism of OxA acting on OxR1 by theOxR1-selective antagonist SB-334867-A, can be detected as a result ofits effect on the signal due to the proximity of RC1 and RC2 modulatedby the modulator, in this case OxA.

Example 4 Use of a Tag on IG3 that Does Not Constitute a ReporterComponent

Referring now to FIG. 7, eBRET signals were measured from cellstransiently co-expressing TRHR/Rluc and EGFP/barr1 or EGFP/barr2 witheither pcDNA3 or HA-OxR2. Ligand treatments were either OxA or TRH only.

Prior to ligand treatment (added at 0 minutes), baseline eBRET signalswere recorded for each of the receptor combinations. PBS treated cellsexpressing each of the combinations exhibited only baseline eBRETsignals for the entire recording period (80 minutes) (data not shown).Following treatment with OxA, cells expressing HA-OxR2 and either of theEGFP/barrs exhibited an eBRET signal reaching a plateau after more than10 minutes (EGFP/barr1, black diamonds and EGFP/barr2, black circles).In cells expressing TRHR/Rluc only (no HA-OxR2), TRH stimulated a rapidincrease in eBRET signal reaching a peak in the first few minutes, thesignal then drifted down slightly over the remainder of the recordingperiod (grey squares). No increase in eBRET signal above baseline wasobserved following OxA addition to cells lacking HA-OxR2 (greytriangles).

This example shows a signal resulting from the proximity of RC1 and RC2detected for the combination where the thyrotropin releasing hormonereceptor (TRHR) is IG1, Rluc is RC1, beta-arrestin 1 (barr1) orbeta-arrestin 2 (barr2) is IG2, EGFP is RC2 and hemagglutin (HA)epitope-tagged OxR2 (HA-OxR2) is IG3 when the modulator, OxA, modulatesthe association of IG2 and IG3 as a result of interacting specificallywith IG3.

This example demonstrates that IG3 can be tagged, such as by theaddition of a hemagglutin (HA) epitope-tag, however, this tag does notconstitute a reporter component and does not interfere with and/orcontribute to the signal generated by the proximity of RC1 and RC2. Suchtagging enables additional information to be ascertained, such as therelative expression level of IG3.

Example 5 Use of a Mutant Beta-Arrestin as an Interacting Group

Referring now to FIG. 8, eBRET signals were measured from cellstransiently co-expressing TRHR/Rluc and EGFP/barr1 or EGFP/barr1phosphorylation-independent mutant R169E (EGFP/barr1R169E) with eitherpcDNA3 or OxR2. Ligand treatments were either OxA or TRH only.

Prior to ligand treatment (added at 0 minutes), baseline eBRET signalswere recorded for each of the receptor combinations. PBS treated cellsexpressing each of the combinations exhibited only baseline eBRETsignals for the entire recording period (100 minutes) (white squares,white diamonds and black diamonds). Following treatment with OxA, cellsexpressing OxR2 and either EGFP/barr1 (black circles) or EGFP/barr1R169E(black triangles) exhibited an eBRET signal with the EGFP/barr1 reachinga plateau after more than 10 minutes while the EGFP/barr1R169E showed alower signal which reached a plateau by 20 minutes. In cells expressingTRHR/Rluc only (no OxR2) with either of the barrs, TRH stimulated arapid increase in eBRET signal reaching a peak in the first few minutes,the signal then drifted down slightly over the remainder of therecording period. The signal for the EGFP/barr1R169E (white triangles)was lower than that for EGFP/barr1 (white circles), which may reflectlower expression levels of this protein.

This example shows a signal resulting from the proximity of RC1 and RC2detected for the combination where the thyrotropin releasing hormonereceptor (TRHR) is IG1, Rluc is RC1, barr1 or barr1R169E is IG2, EGFP isRC2 and OxR2 is IG3.

This example demonstrates that a detectable signal can be generated whenusing a mutant beta-arrestin, such as the beta-arrestin 1phosphorylation-independent mutant R169E, as one of the interactinggroups.

Example 6 Measurement of a Detectable Signal Indicative of the MolecularAssociation of the C-Terminally Truncated Thyrotropin Releasing HormoneReceptor with the Orexin Receptor

Referring now to FIG. 9, eBRET signals were measured from cellstransiently co-expressing TRHR335/Rluc and EGFP/barr1 with either OxR2or TRHR. Ligand treatments were either OxA or TRH only.

Prior to ligand treatment (added at 0 minutes), baseline eBRET signalswere recorded for each of the receptor combinations. Following treatmentwith OxA, cells expressing OxR2 (black circles) exhibited an eBRETsignal reaching a plateau after about 20 minutes. In contrast, no eBRETsignal above baseline was observed from cells expressing TRHR whentreated with OxA (white triangles), or from cells expressing OxR2 whentreated with TRH (black squares).

This example shows a signal resulting from the proximity of RC1 and RC2detected for the combination where the thyrotropin releasing hormonereceptor truncated at amino acid 335 (TRHR335) is IG1, Rluc is RC1,beta-arrestin 1 (barr1) is IG2, EGFP is RC2 and OxR2 or TRHR is IG3.

This example demonstrates that a detectable signal can be generated whenIG1 does not interact with IG2, in this case, a truncated TRHR that doesnot interact with barr1 (Heding et al. (2000) Endocrinology 141,299-306). The lack of signal observed in FIG. 9 upon treatment ofTRHR335/Rluc+EGFP/barr1+OxR2 with TRH confirms that the signal observedupon OxA treatment of this agent combination is not due to IG1-IG2association.

Therefore, this example provides further and distinct evidence for themolecular association of the thyrotropin releasing hormone receptor withthe orexin receptor, as the inability of IG1 to interact with IG2 isindicative of RC1 and RC2 proximity as a result of IG2-IG3-IG1association and not IG1-IG2 association. This provides further evidenceagainst signals originating from non-specific IG1-IG2 association in theabsence of an IG1-specific ligand.

Furthermore, this example demonstrates that the signal results fromIG2-IG3-IG1 association as opposed to IG3 activation causingtransactivation of IG1, which then associates with IG2, thereby bringingRC1 and RC2 into close proximity without IG2 and IG3 associating.

Example 7 Measurement of a Detectable Signal in a CharacteristicDose-Dependent Manner Indicative of the Molecular Association of TRHRwith OxR2

Referring now to FIGS. 10, 11 and 12, BRET signals were measured fromcells transiently co-expressing: TRHR/Rluc and barr2/Venus with pcDNA3(treated with increasing doses of TRH; FIG. 10); OxR2/Rluc andbarr2/Venus with pcDNA3 (treated with increasing doses of OxA; FIG. 11);and TRHR/Rluc and barr2/Venus with OxR2 (treated with increasing dosesof OxA; FIG. 12).

This example shows: a TRH dose-response curve for TRHR as IG1, Rluc asRC1, barr2 as IG2, Venus as RC2 and in the absence of IG3 (FIG. 10); anOxA dose-response curve for OxR2 as IG1, Rluc as RC1, barr2 as IG2,Venus as RC2 and in the absence of IG3 (FIG. 11); and OxA dose-responsecurves for the TRHR as IG1, Rluc as RC1, barr2 as IG2, Venus as RC2 andOxR2 as IG3 (FIG. 12).

This example demonstrates that signals can be detected in adose-dependent manner. Furthermore, the EC₅₀ values for signalsresulting from the modulator (OxA) acting on IG3 (OxR2) and consequentproximity of IG1-RC1 (TRHR/Rluc) and IG2-RC2 (barr2/Venus; FIG. 12) arecomparable to those from OxA activation of IG1 (OxR2) resulting inproximity of IG1-RC1 (OxR2/Rluc) and IG2-RC2 (barr2/Venus; FIG. 11), anddistinct from those from TRH activation of IG1 (TRHR) resulting inproximity of IG1-RC1 (TRHR/Rluc) and IG2-RC2 (barr2/Venus; FIG. 10).

Therefore, this example further demonstrates that the signal resultsfrom IG2-IG3-IG1 association as opposed to IG1-IG2 association.

The dose-response Hill slopes for OxA activation of IG1 (OxR2) resultingin proximity of IG1-RC1 (OxR2/Rluc) and IG2-RC2 (barr2/Venus; FIG. 11);and TRH activation of IG1 (TRHR) resulting in proximity of IG1-RC1(TRHR/Rluc) and IG2-RC2 (barr2/Venus; FIG. 10) are both approximately 1.In contrast, the dose-response Hill slopes for modulator (OxA) acting onIG3 (OxR2) resulting in proximity of IG1-RC1 (TRHR/Rluc) and IG2-RC2(barr2/Venus; FIG. 12) are substantially greater than 1.

Therefore, this example demonstrates the potential for identifying andmonitoring specific molecular associations using the Hill slope as anindicator.

This example further demonstrates that different forms of Rluc substrate(reporter component initiator), in this case coelenterazine h andEnduRen, can be used to generate data with similar EC₅₀ values (FIG.12).

Example 8 Measurement of Additive Detectable Signals in a Dose-DependentManner Indicative of the Molecular Association of TRHR With OxR2

Referring now to FIGS. 13 and 14, BRET signals were measured from cellstransiently co-expressing TRHR/Rluc and EGFP/barr1 in the absence ofOxR2 with increasing doses of TRH, as well as cells transientlyco-expressing TRHR/Rluc and EGFP/barr1 with OxR2 with increasing dosesof OxA with and without 10⁻⁶M TRH, or increasing doses of TRH with 10⁻⁶MOxA.

This example shows a curve mathematically generated by addition of theligand-induced signal generated with 10⁻⁶M TRH (from the TRH:TRHR/Rluc+EGFP/barr1 curve) to each of the points generated for the OxA:TRHR/Rluc+EGFP/barr1+OxR2 curve (TRHR/Rluc+EGFP/barr1+OxR2: TRH(10⁻⁶M)+OxA: Data calculated) overlain on a curve generated from dataobserved for the TRHR/Rluc+EGFP/barr1+OxR2: TRH (10⁻⁶M)+OxA combination(FIG. 13).

Furthermore, this example shows a curve mathematically generated byaddition of the ligand-induced signal generated with 10⁻⁶M OxA (from theOxA: TRHR/Rluc+EGFP/barr1+OxR2 curve) to each of the points generatedfor the TRH: TRHR/Rluc+EGFP/barr1 curve (TRHR/Rluc+EGFP/barr1+OxR2:TRH+OxA (10⁻⁶M): Data calculated) overlain on a curve generated fromdata observed for the TRHR/Rluc+EGFP/barr1+OxR2: TRH+OxA (10⁻⁶M)combination (FIG. 14).

Therefore, this example clearly demonstrates the additive effect ofcombined treatment with IG1 ligand (TRH) and IG3 ligand (OxA; modulator)in a dose dependent manner.

Therefore, this example provides further evidence for the molecularassociation of the thyrotropin releasing hormone receptor with theorexin receptor, as this additive effect is indicative of RC1 and RC2proximity as a result of IG1-IG2 association in addition to IG2-IG3-IG1association. This provides further evidence against signals originatingfrom non-specific IG1-IG2 association in the absence of an IG1-specificligand. Without wishing to be bound by theory, this additive effect mayalso be partly due to IG1 ligand acting as a modulator to modulate theassociation of IG2 and IG3 via allosteric effects on IG3. Furthermore,this additive effect may also be partly due to an active IG conformation(one that is bound to agonist) being more favourable for signalgeneration, perhaps enabling increased proximity of RC1 and RC2, or morefavourable relative orientation of RC1 and RC2.

Example 9 Measurement of Additive Detectable Signals in a Dose-DependentManner Indicative of the Molecular Association of TRHR335 with OxR2

Referring now to FIG. 15, BRET signals were measured from cellstransiently co-expressing TRHR335/Rluc, barr2/Venus and OxR2 withincreasing doses of TRH and OxA alone or in combination.

This example demonstrates, using dose response curves, that TRH additiondoes not result in a BRET signal due to RC1 (Rluc) and RC2 (Venus)proximity as a result of interacting with IG1 (TRHR335) when IG1(TRHR335) is not able to interact with IG2 (barr2). However, a BRETsignal due to RC1 (Rluc) and RC2 (Venus) proximity as a result ofinteracting with IG3 (OxR2) is observed, indicating an association ofIG1 (TRHR335) and IG3 (OxR2). This confirms the data in example 6.

This example further shows that, despite the lack of BRET signalresulting from TRH addition, an increased signal above that observedwith OxA addition alone is observed upon addition of both TRH and OxA.

This demonstrates that activation of IG1 (TRHR335) does influence signalgeneration, despite not being able to contribute to IG1-IG2(TRHR335-barr2) association. Without wishing to be bound by theory, thismay imply that IG1 is influencing IG3 by an allosteric mechanism. Thismay also imply that an active IG conformation (one that is bound toagonist) is more favourable for signal generation, perhaps enablingincreased proximity of RC1 and RC2, or more favourable relativeorientation of RC1 and RC2.

Therefore, this example further demonstrates that co-treatment of IG1and IG3 can result in additional signal generation and/or informationcompared to treatment of IG3 alone and that such co-treatment isencompassed by the present invention.

Example 10 Measurement of a Detectable Signal Indicative of theMolecular Association of TRHR with OxR2 at Various Expression Levels

Referring now to FIG. 16, eBRET signals were measured from cellstransiently co-expressing TRHR/Rluc, EGFP/barr1 and OxR2 followingaddition of 10⁻⁶M OxA.

This example shows cumulative eBRET reads over time for each combinationof receptors (IG1 and IG3; data captured over 83 mins). The same amountof EGFP/barr1 (IG2-RC2) is transfected for each experiment. TRHR/Rluc(IG1-RC1) is transfected at a constant amount (0.1 μg DNA/well) whileOxR2 (IG3) is transfected at varying amounts of DNA (0, 0.01, 0.05, 0.1,0.5, 0.7 μg DNA/well). The signal is only detected when OxR2 (IG3) isexpressed (no signal was recorded at 0 μg OxR2).

This example demonstrates that signal can be detected when DNAconcentrations of OxR2 are as low as 0.01 μg DNA/well.

Furthermore, this example demonstrates that increasing the amounts ofOxR2 DNA in each transfection results in increases in the detectablesignal. The largest detectable signal is observed at a 1:1 ratio of DNAconcentration (0.1:0.1 μg DNA/well). Further increases in the OxR2 DNAconcentration (0.5 or 0.7 μg DNA/well) with levels higher than theamount of TRHR/Rluc DNA results in a lower signal being detected.

This example implies that increasing the number of IG3 molecules (OxR2)leads to a point being reached beyond which the number of IG1 molecules(TRHR) becomes limiting for the formation of hetero-dimers/-oligomers.Consequently, there would be increasing propensity for IG3 molecules(OxR2) not associated with IG1 molecules (TRHR) to associate withIG2-RC2 (EGFP/barr1) upon interacting with the modulator (OxA) without asignal being generated. Therefore, signal generation would be inhibiteddue to the competition for IG2-RC2 (EGFP/barr1) association.

Therefore, this example provides further and distinct evidence for themolecular association of the thyrotropin releasing hormone receptor withthe orexin receptor, as such decreases in signal with increases in IG3concentration beyond that of IG1 concentration would not be expected tooccur if the signal was not dependent upon specific molecularassociation of IG1 and IG3.

Example 11 Measurement of a Detectable Signal Indicative of theMolecular Association of TRHR with OxR2 in 384-Well Plates

Referring now to FIG. 17, BRET signals were measured from cellstransiently co-expressing TRHR/Rluc, barr2/Venus and OxR2 withincreasing doses of OxA in 96-well and 384-well microplates.

BRET measurements were carried out using the same concentration of cellsexpressing the same concentration of agents, the same concentration ofRluc substrate (reporter component initiator) and the same concentrationof ligand (modulator). The total volume added to each well of the384-well plate was approximately half that added to each well of the96-well plate.

This example demonstrates measurement of a detectable signal indicativeof the molecular association of TRHR with OxR2 in a dose-dependentmanner in 384-well plates in addition to 96-well plates.

Therefore, this example demonstrates that the method described in theinvention is able to be scaled down, thereby making it amenable tohigh-throughput screening applications.

Example 12 Measurement of a Detectable Signal Indicative of theMolecular Association of the Thyrotropin Releasing Hormone Receptor asIG3 with the Orexin Receptor as IG1

Referring now to FIG. 18, eBRET signals were measured from cellstransiently co-expressing OxR2/Rluc8 and barr2/Venus either with HA-TRHRor pcDNA3. Ligand treatments were either OxA or TRH.

Prior to ligand or vehicle treatment (added at 0 minutes), a baselineeBRET signal was recorded for each of the receptor combinations. Withinthe first 5 minutes, OxA treatment of cells co-expressing OxR2/Rluc8 andbarr2/Venus with HA-TRHR, resulted in the eBRET signal rapidly reachinga peak of 0.1 and this signal remained high for the entire recordingperiod (over an hour). A signal was also observed following TRHtreatment of cells co-expressing OxR2/Rluc8, barr2/Venus and HA-TRHR.This signal however gradually increased over time to reach 0.05. Noligand-induced eBRET signal was observed following TRH treatment ofcells co-expressing OxR2/Rluc8 and barr2/Venus with pcDNA3.

This example demonstrates that a signal resulting from the proximity ofRC1 and RC2 is detected specifically for the combination where OxR2 isIG1, Rluc8 is RC1, beta-arrestin 2 (barr2) is IG2, Venus is RC2 andHA-TRHR is IG3, and when the modulator, in this case TRH, modulates theassociation of IG2 and IG3 as a result of interacting specifically withIG3.

This example demonstrates that the molecular association of thethyrotropin releasing hormone receptor with the orexin receptor isdetected with the thyrotropin releasing hormone receptor as IG3 and theorexin receptor as IG1. This demonstrates detection of the molecularassociation of these receptors using an alternative arrangement of IG'scompared to previous examples.

This example also demonstrates the use of a second type of luciferase,Rluc8, which in this case is used as RC1 with Venus as RC2.

This example further demonstrates that the alternative method ofcalculating the eBRET signal described in Pfleger et al., 2006 (CellSignal 18, 1664-1670) and Pfleger et al., 2006 (Nat Protoc 1, 336-344)can be used in the measurement of a detectable signal indicative of themolecular association of the thyrotropin-releasing hormone receptor andthe orexin receptor.

As in example 4, this example demonstrates that IG3 can be tagged, suchas by the addition of a hemagglutin (HA) epitope-tag, however, this tagdoes not constitute a reporter component and does not interfere withand/or contribute to the signal generated by the proximity of RC1 andRC2. Such tagging enables additional information to be ascertained, suchas the relative expression level of IG3.

Example 13 Measurement of a Detectable Signal Indicative of theMolecular Association of the Thyrotropin Releasing Hormone Receptor withthe Orexin Receptor with a Z-Factor in Excess of 0.6

Referring now to FIGS. 19, 20 and 21, eBRET signals were measured fromcells transiently co-expressing TRHR/Rluc8 and barr2/Venus with HA-OxR2aliquoted into all wells of a 96-well plate. Phosphate-buffered saline(PBS) was added to the first two rows and the last two rows of the96-well plate (48 wells in total) as a vehicle control. OxA was added tothe middle four rows of the 96-well plate (48 wells in total). Data arepresented as fluorescence/luminescence.

Prior to ligand or vehicle treatment (added at 0 minutes), baselinereadings were recorded. OxA treatment of cells co-expressing TRHR/Rluc8and barr2/Venus with HA-OxR2 resulted in an increase in thefluorescence/luminescence ratio (FIG. 20) that was not observedfollowing treatment with phosphate-buffered saline (PBS) vehicle control(FIG. 19). Analysis of the fluorescence/luminescence ratios comparing48-wells treated with OxA (defined as ‘signal’ with respect to z-factorcalculation) and 48-wells treated with PBS (defined as ‘background’ withrespect to z-factor calculation) results in a z-factor of 0.67 using thecalculation described by Zhang et al., 1999 (J Biomol Screen 4, 67-73).Means are shown as solid lines and 3 standard deviations from the meanare shown as dotted lines.

This example demonstrates that a signal resulting from the proximity ofRC1 and RC2 is detected specifically for the combination where TRHR isIG1, Rluc8 is RC1, beta-arrestin 2 (barr2) is IG2, Venus is RC2 andHA-OxR2 is IG3, and when the modulator, in this case OxA, modulates theassociation of IG2 and IG3 as a result of interacting specifically withIG3.

This example demonstrates that the molecular association of thethyrotropin releasing hormone receptor with the orexin receptor isdetected in a manner that results in a z-factor in excess of 0.6 and istherefore amenable to high-throughput screening.

This example further demonstrates a third method of representing BRETdata that can be used in representing a detectable signal indicative ofthe molecular association of the thyrotropin-releasing hormone receptorand the orexin receptor.

As in examples 4 and 12, this example demonstrates that IG3 can betagged, such as by the addition of a hemagglutin (HA) epitope-tag,however, this tag does not constitute a reporter component and does notinterfere with and/or contribute to the signal generated by theproximity of RC1 and RC2. Such tagging enables additional information tobe ascertained, such as the relative expression level of IG3.

1. A hetero-dimeric or hetero-oligomeric receptor, comprising at leastone thyrotropin releasing hormone receptor subunit associated with atleast one orexin receptor subunit.
 2. A method for the treatment of apatient suffering from an orexin-related ailment by administering atherapeutically effective amount of a thyrotropin-releasing hormonereceptor agonist, inverse agonist, partial agonist or antagonist.
 3. Amethod according to claim 2, wherein the thyrotropin-releasing hormonereceptor agonist, inverse agonist or antagonist is co-administered withan orexin receptor agonist, inverse agonist, partial agonist orantagonist.
 4. A method for the treatment of a patient suffering from athyrotropin-releasing hormone-related ailment by administering atherapeutically effective amount of an orexin receptor agonist, inverseagonist, partial agonist or antagonist.
 5. A method according to claim4, wherein the orexin receptor agonist, inverse agonist or antagonist isco-administered with a thyrotropin-releasing hormone receptor agonist,inverse agonist, partial agonist or antagonist.
 6. A method for themanufacture of a medicament for the treatment of a patient sufferingfrom an orexin-related ailment, said medicament comprising atherapeutically effective amount of a thyrotropin releasing hormonereceptor agonist, inverse agonist, partial agonist or antagonist.
 7. Themethod according to claim 6, wherein the medicament further comprises anorexin receptor agonist, inverse agonist, partial agonist or antagonist.8. A method for the manufacture of a medicament for the treatment of apatient suffering from a thyrotropin-releasing hormone-related ailment,said medicament comprising a therapeutically effective amount of anorexin receptor agonist, inverse agonist, partial agonist or antagonist.9. The A method according to claim 8, wherein the medicament furthercomprises a thyrotropin-releasing hormone receptor agonist, inverseagonist, partial agonist or antagonist.
 10. A method for the manufactureof a medicament for the treatment of a patient suffering from anorexin-related ailment said medicament comprising a therapeuticallyeffective amount of a thyrotropin-releasing hormone-selective bindingagent, or fragment thereof.
 11. The method according to claim 10,wherein the thyrotropin-releasing hormone-selective binding agent is anantibody, a humanised antibody, a polyclonal antibody, a monoclonalantibody, a chimeric antibody, a CDR-grafted antibody or ananti-idiotypic antibody.
 12. A method for the manufacture of amedicament for the treatment of a patient suffering from athyrotropin-releasing hormone-related ailment, said medicamentcomprising a therapeutically effective amount of an orexin-selectivebinding agent, or fragment thereof.
 13. The method according to claim12, wherein the orexin-selective binding agent is an antibody, ahumanised antibody, a polyclonal antibody, a monoclonal antibody, achimeric antibody, a CDR-grafted antibody or an anti-idiotypic antibody.14. A method for screening a test compound for thyrotropin releasinghormone receptor/orexin receptor hetero-dimer/-oligomer selectiveactivity, the method comprising the steps of: a) determining whether,and/or the extent to which, the test compound interacts with the orexinreceptor while the orexin receptor is associated with the thyrotropinreleasing hormone receptor; and b) if the test compound interacts withthe orexin receptor while the orexin receptor is associated with thethyrotropin releasing hormone receptor, determining whether, or theextent to which the test compound interacts with the orexin receptor inthe absence of the thyrotropin releasing hormone receptor; such that thetest compound that exhibits greater affinity and/or potency and/orefficacy when interacting with the orexin receptor while the orexinreceptor is associated with the thyrotropin releasing hormone receptoris selective for the thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer.
 15. A method for screening a testcompound for thyrotropin releasing hormone receptor/orexin receptorhetero-dimer/-oligomer selective activity, the method comprising thesteps of: a) determining whether, and/or the extent to which, the testcompound interacts with the thyrotropin releasing hormone receptor whilethe thyrotropin releasing hormone receptor is associated with the orexinreceptor; and b) if the test compound interacts with the thyrotropinreleasing hormone receptor while the thyrotropin releasing hormonereceptor is associated with the orexin receptor, determining whether, orthe extent to which the test compound interacts with the thyrotropinreleasing hormone receptor in the absence of the orexin receptor; suchthat the test compound that exhibits greater affinity and/or potencyand/or efficacy when interacting with the thyrotropin releasing hormonereceptor while the thyrotropin releasing hormone receptor is associatedwith the orexin receptor is selective for the thyrotropin releasinghormone receptor/orexin receptor hetero-dimer/-oligomer.
 16. A methodfor screening a test compound for thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer selective antagonism orpartial agonism, the method comprising the steps of: determiningwhether, and/or the extent to which, the test compound is an antagonistor partial agonist of the thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer, by contacting said test compound with asystem comprising: i) a first agent, comprising the orexin receptorcoupled to a first reporter component; ii) a second agent, comprising aninteracting group coupled to a second reporter component; iii) a thirdagent, comprising the thyrotropin releasing hormone receptor; iv) anagonist of the orexin receptor, the thyrotropin releasing hormonereceptor and/or the thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer; wherein proximity of the first andsecond reporter components generates a signal; and wherein the modulatormodulates the association of the interacting group with the thyrotropinreleasing hormone receptor; b) detecting a decrease in the signal as adetermination of whether and/or the extent to which the test compound isan antagonist or partial agonist of the thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer; and c) if the testcompound is an antagonist or partial agonist of the thyrotropinreleasing hormone receptor/orexin receptor hetero-dimer/-oligomer,determining whether, or the extent to which the test compound is anantagonist or partial agonist of the thyrotropin releasing hormonereceptor in the absence of the orexin receptor and the orexin receptorin the absence of the thyrotropin releasing hormone receptor; such thata test compound that exhibits greater antagonistic or partial agonisticproperties when interacting with the thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer is selective for thethyrotropin releasing hormone receptor/orexin receptorhetero-dimer/-oligomer.
 17. A method for screening a test compound forthyrotropin releasing hormone receptor/orexin receptorhetero-dimer/-oligomer selective antagonism or partial agonism, themethod comprising the steps of: a) determining whether, and/or theextent to which, the test compound is an antagonist or partial agonistof the thyrotropin releasing hormone receptor/orexin receptorhetero-dimer/-oligomer, by contacting said test compound with a systemcomprising: i) a first agent, comprising the thyrotropin releasinghormone receptor coupled to a first reporter component; ii) a secondagent, comprising an interacting group coupled to a second reportercomponent; iii) a third agent, comprising the orexin receptor; and iv)an agonist of the orexin receptor, the thyrotropin releasing hormonereceptor and/or the thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer; wherein proximity of the first andsecond reporter components generates a signal; and wherein the modulatormodulates the association of the interacting group with the orexinreceptor; b) detecting a decrease in the signal as a determination ofwhether and/or the extent to which the test compound is an antagonist orpartial agonist of the thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer; and c) if the test compound is anantagonist or partial agonist of the thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer, determining whether, orthe extent to which the test compound is an antagonist or partialagonist of the thyrotropin releasing hormone receptor in the absence ofthe orexin receptor and the orexin receptor in the absence of thethyrotropin releasing hormone receptor; such that a test compound thatexhibits greater antagonistic or partial agonistic properties wheninteracting with the thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer is selective for the thyrotropinreleasing hormone receptor/orexin receptor hetero-dimer/-oligomer.
 18. Amethod for screening a test compound for thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer selective inverseagonism, the method comprising the steps of: a) determining whether,and/or the extent to which, the test compound is an inverse agonist ofthe thyrotropin releasing hormone receptor/orexin receptorhetero-dimer/-oligomer, by contacting said test compound with a systemcomprising: i) a first agent, comprising the orexin receptor coupled toa first reporter component; ii) a second agent, comprising aninteracting group coupled to a second reporter component; and iii) athird agent, comprising a constitutively active thyrotropin releasinghormone receptor; wherein proximity of the first and second reportercomponents generates a signal; and wherein the modulator modulates theassociation of the interacting group with the thyrotropin releasinghormone receptor; b) detecting a decrease in the signal as adetermination of whether and/or the extent to which the test compound isan inverse agonist of the thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer; and c) if the test compound is aninverse agonist of the thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer, determining whether, or the extent towhich the test compound is an inverse agonist of the thyrotropinreleasing hormone receptor in the absence of the orexin receptor and theorexin receptor in the absence of the thyrotropin releasing hormonereceptor; such that a test compound that exhibits greater inverseagonistic properties when interacting with the thyrotropin releasinghormone receptor/orexin receptor hetero-dimer/-oligomer is selective forthe thyrotropin releasing hormone receptor/orexin receptorhetero-dimer/-oligomer.
 19. A method for screening a test compound forthyrotropin releasing hormone receptor/orexin receptorhetero-dimer/-oligomer inverse agonism, the method comprising the stepsof: a) determining whether, and/or the extent to which, the testcompound is an inverse agonist of the thyrotropin releasing hormonereceptor/orexin receptor hetero-dimer/-oligomer, by contacting said testcompound with a system comprising: i) a first agent, comprising thethyrotropin-releasing hormone receptor coupled to a first reportercomponent; ii) a second agent, comprising an interacting group coupledto a second reporter component; iii) a third agent, comprising aconstitutively active orexin receptor; and wherein proximity of thefirst and second reporter components generates a signal; and wherein themodulator modulates the association of the interacting group with theorexin receptor; b) detecting a decrease in the signal as adetermination of whether and/or the extent to which the test compound isan inverse agonist of the thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer; and c) if the test compound is aninverse agonist of the thyrotropin releasing hormone receptor/orexinreceptor hetero-dimer/-oligomer, determining whether, or the extent towhich the test compound is an inverse agonist of the thyrotropinreleasing hormone receptor in the absence of the orexin receptor and theorexin receptor in the absence of the thyrotropin releasing hormonereceptor; such that a test compound that exhibits greater inverseagonistic properties when interacting with the thyrotropin releasinghormone receptor/orexin receptor hetero-dimer/-oligomer is selective forthe thyrotropin releasing hormone receptor/orexin receptorhetero-dimer/-oligomer.
 20. Selective agonists and/or antagonists and/orinverse agonists and/or partial agonists of the a thyrotropin releasinghormone receptor/orexin receptor hetero-dimer/-oligomer.
 21. A methodfor the treatment of a patient suffering from a thyrotropin-releasinghormone-related ailment by administering a therapeutically effectiveamount of a selective orexin receptor/thyrotropin-releasing hormonereceptor hetero-dimer/-oligomer agonist, inverse agonist, partialagonist or antagonist.
 22. A method according to claim 21, wherein theselective orexin receptor/thyrotropin-releasing hormone receptorhetero-dimer/-oligomer agonist, inverse agonist, partial agonist orantagonist is co-administered with a thyrotropin-releasing hormonereceptor agonist, inverse agonist, partial agonist or antagonist.
 23. Amethod according to claim 21, wherein the selective orexinreceptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomeragonist, inverse agonist, partial agonist or antagonist isco-administered with an orexin receptor agonist, inverse agonist,partial agonist or antagonist.
 24. A method for the treatment of apatient suffering from a orexin-related ailment by administering atherapeutically effective amount of a selective orexinreceptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomeragonist, inverse agonist, partial agonist or antagonist.
 25. A methodaccording to claim 24, wherein the selective orexinreceptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomeragonist, inverse agonist, partial agonist or antagonist isco-administered with a thyrotropin-releasing hormone receptor agonist,inverse agonist, partial agonist or antagonist.
 26. A method accordingto claim 24, wherein the selective orexin receptor/thyrotropin-releasinghormone receptor hetero-dimer/-oligomer agonist, inverse agonist,partial agonist or antagonist is co-administered with an orexin receptoragonist, inverse agonist, partial agonist or antagonist.
 27. A methodfor the manufacture of a medicament for the treatment of a patientsuffering from a thyrotropin-releasing hormone-related ailmentcomprising use of a therapeutically effective amount of a selectiveorexin receptor/thyrotropin-releasing hormone receptorhetero-dimer/-oligomer agonist, inverse agonist, partial agonist orantagonist.
 28. A method according to claim 27, wherein the medicamentcontains an orexin receptor agonist, inverse agonist, partial agonist orantagonist.
 29. A method according to claim 27, wherein medicamentcontains a thyrotropin-releasing hormone receptor agonist, inverseagonist, partial agonist or antagonist.
 30. A method for the manufactureof a medicament for the treatment of a patient suffering from anorexin-related ailment comprising use of a therapeutically effectiveamount of a selective orexin receptor/thyrotropin-releasing hormonereceptor hetero-dimer/-oligomer agonist, inverse agonist, partialagonist or antagonist.
 31. A method according to claim 30, wherein themedicament contains an orexin receptor agonist, inverse agonist, partialagonist or antagonist.
 32. A method according to claim 30, wherein themedicament contains a thyrotropin-releasing hormone receptor agonist,inverse agonist, partial agonist or antagonist.